Bid, a BH-3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH-3 domain which then induces apoptosis by interacting with proapoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid, and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. BidS contains the N-terminal regulatory domains of Bid without the BH-3 domain; BidEL corresponds to full length Bid with additional N-terminal sequence; and BidES contains only the Bid sequence downstream of the BH-3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies BidEL induces apoptosis whereas BidS abrogates the proapoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. BidES induces apoptosis, but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These 3 novel, endogenously expressed isoforms of Bid are distinct in their expression, cellular localization and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage, and thus influence the fate of cells exposed to a range of proapoptotic stimuli.