Zimbalist was most widely known for his starring role as Inspector Lewis Erskine in the Quinn Martin television production, The F.B.I., which premiered on September 19, 1965 and ended with its final episode on September 8, 1974. Zimbalist was generous in his praise of producer Martin and of his own experience starring in the show. Those who worked with him on the show were equally admiring of the star's professionalism and likable personality.

Zimbalist maintained a strong personal relationship with J. Edgar Hoover, who requested technical accuracy for the show and that agents be portrayed in the best possible light. Actors who played F.B.I. employees were required by Hoover to undergo a background check. Zimbalist passed his background check with ease. He subsequently spent a week in Washington, D.C., where he was interviewed by Hoover, and at the F.B.I. Academy in Quantico, Virginia. Hoover and Zimbalist remained mutual admirers for the rest of Hoover's life.

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