The striking similarities between the three-dimensional structures of pro-survival proteins, Bax/Bak and certain BH3-only proteins such as Bid, raises the conundrum of what dictates their opposing functions. Here, we describe key sequence signatures within the BH3 domains of these divergent proteins that contribute to their abilities to promote either cell survival or cell death. Accordingly, through mutagenesis studies, we were able to convert pro-survival protein BH3 sequences into potent killers of cells whilst introduction of the complementary mutations into Bak negates its cell killing activity. Analysis of the binding affinities of the different mutant sequences established a biochemical basis for the disparate activities, whilst a new X-ray crystallographic structure provides further insights into the different behaviours of the pro-apoptotic and pro-survival BH3 sequences.