Hartley began her career as a 13-year-old in the White Barn Theatre in Norwalk, Connecticut. In her teens as a stage actress, she was coached and mentored by Eva Le Gallienne. She graduated from Westport’s Staples High School in 1957, where she was an active member of the school’s theater group, Staples Players. Hartley also worked at the American Shakespeare Festival.
Hartley’s first credited film appearance was alongside Randolph Scott and Joel McCrea in the 1962 Sam Peckinpah western Ride the High Country; the role earned her a BAFTAnomination. She continued to appear in film during the 1960s, including the lead role in the adventure Drums of Africa (1963), and prominent supporting roles in Alfred Hitchcock‘s psychological thriller Marnie (1964) — alongside Tippi Hedren and Sean Connery — and the John Sturges drama Marooned (1969).
Hartley also guest starred in numerous TV series during the decade, with appearances in Gunsmoke, The Twilight Zone (the episode “The Long Morrow“), The Travels of Jaimie McPheeters (starring a young Kurt Russell), the syndicated Death Valley Days (then hosted by Ronald Reagan),Judd, for the Defense, Bonanza and Star Trek among others. In 1965, she had a significant role as Dr. Claire Morton in 32 episodes of Peyton Place.With Dennis Weaver in Gunsmoke(1962)
Hartley continued to feature in numerous film and TV roles during the 1970s, including appearances in two Westerns alongside Lee Van Cleef, Barquero (1970) and The Magnificent Seven Ride (1972), as well as landing guest roles in episodes of series including Emergency, McCloud, Little House on the Prairie, Police Woman and Columbo — starring in two editions of the latter alongside Peter Falk; Publish or Perish co-starring Jack Cassidy (1974) and Try and Catch Me with Ruth Gordon (1977). Hartley portrays similar characters as a publisher’s assistant in both episodes.
Her role as psychologist Dr. Carolyn Fields in “Married”, a 1978 episode of the TV series The Incredible Hulk — in which she marries Bill Bixby‘s character, the alter ego of the Hulk — won Hartley the Primetime Emmy Award for Outstanding Lead Actress in a Drama Series. She would be nominated for the same award for her performance in an episode of The Rockford Files the following year.
In 1983, Hartley reunited with Bixby in the sitcom Goodnight, Beantown, which ran for two seasons; the role earned her yet another Emmy Award nomination. (She would later work alongside Bixby again in the 1992 TV movie A Diagnosis of Murder, the first of three TV movies that would launch the series Diagnosis: Murder).
In the 1990s, Hartley toured with Elliott Gould and Doug Wert in the revival of the mystery play Deathtrap. Numerous roles in TV movies and guest appearances in TV series during the 1990s and 2000s would follow, including Murder, She Wrote (1992), Courthouse (1995), Nash Bridges (2000) and NCIS (2005). She had recurring roles as Sister Mary Daniel in the soap opera One Life to Live (1999–2001; 10 episodes), and as Lorna Scarry in 6 episodes of Law & Order: Special Victims Unit (2003–2011).
From 1995 to 2015, she hosted the long-running television documentary series Wild About Animals, an educational program.
In 2006, Hartley starred in her own one-woman show, If You Get to Bethlehem, You’ve Gone Too Far, which ran in Los Angeles. She returned to the stage in 2014 as Eleanor of Aquitaine with Ian Buchanan‘s Henry in the Colony Theater Company production of James Goldman‘s The Lion in Winter.