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Born in then-Jersualem, Palestine (in current day Israel), Persoff's father was silversmith and painter. While initially interested in painting, he saw that the artists of 1936 struggled to make paining a livelihood and thus pursued acting.
As an actor, he began on the New York stage as "Galileo" starring Charles Laughton and, while still working on Broadway, later appeared in various film and television productions. His first foray into film was a brief appearance in 1948's The Naked City.
His roles became much more prolific, guest starring on various television series from the 1960s to 1990s including genre television such as The Twilight Zone, Voyage to the Bottom of the Sea, Logan's Run, and Star Trek: The Next Generation.
In 1979, he performed an one-man show called "Sholom Aleichem" that played in the United States, Canada and Australia. "Sholom Aleichem" earned Persoff two awards from the Los Angeles Critics Award and the Bay Area Critics Award for best show and best performance that same year.
From 1985 onward, his television appearances became fewer due to his health and high blood pressure, and he took on painting.