Most was born to a Jewish family in Brooklyn, New York City. He lived in Flatbush, Brooklyn, New York, and graduated from Erasmus Hall High School in 1970. He attended Lehigh University for three years from 1970 to 1973, but did not graduate. Originally intending to study engineering, he changed his major to business after his first semester. He made plans during his junior year to spend the summer of 1973 in search of acting jobs in Hollywood before completing his studies at Lehigh. Instead, he landed the Ralph Malph role on his third audition and moved to California to pursue acting full-time.
Most appeared on the 113th and final filmed but never aired episode of Room 222, entitled "Jason and Big Mo", in the role of Louie, the red-headed class wisecracker. David Jolliffe had handled the role of Bernie, the red-headed class wisecracker, for all prior appearances. Room 222 aired for the last time in January 1974, just as Happy Days began, and on the same network, ABC.
Most played jokester Ralph on Happy Days, regularly appearing up until the show’s seventh season and returned in the final season as a guest star. During the first season Most was more of a secondary character, billed only in the end credits, but by the second season he was front billed, as a primary character.
Most has appeared in other film and television work. Film credits include Leo and Loree (1980), EDtv (1999), Planting Melvin (2005), and The Great Buck Howard (2008). He also made the regular round of guest appearances on TV shows like Emergency!, CHiPs, Baywatch, The Love Boat, Sliders, Star Trek: Voyager, Diagnosis: Murder, Yes, Dear, and Glee. He also made an appearance on Charles in Charge, alongside his former Happy Days co-star, Scott Baio. He plays a man who has just won the lottery and, as part of the cameo joke, he runs up to Baio and waving the winning ticket shouts, "It looks like happy days are here again!" (He also receives several looks from Baio that suggests familiarity throughout the episode.) He is sometimes credited as "Donny Most."

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