The N-ethylmaleimide-sensitive fusion protein (NSF) is a cytoplasmic protein implicated in the fusion of intracellular transport vesicles with their target membranes. NSF is thought to function in the fusion of essentially all types of vesicles, including endoplasmic reticulum, Golgi, and endocytic vesicles, as well as secretory vesicles undergoing regulated fusion (for review see Rothman, J.E. (1994) Nature 372, 55-63). However, little experimental evidence exists to address the possibility that organisms might have multiple NSF proteins serving distinct functions in the same or different cells. We previously cloned a neurally expressed Drosophila homolog, dNSF-1 (Ordway, R.W., Pallanck, L., and Ganetzky, B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5715-5719), and have subsequently identified mutations in this gene that confer an apparent failure of synaptic transmission at elevated temperature (Pallanck, L., Ordway, R.W., and Ganetzky, B. (1995) Nature, 376, 25; Siddiqi, O., and Benzer, S. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3253-3257). Here we report that 1) Drosophila contains a second NSF homolog, termed dNSF-2, that exhibits 84% amino acid identity to dNSF-1, 2) dNSF-1 and dNSF-2 display overlapping but different temporal expression, and 3) multiple transcripts are derived from the dNSF-2 gene. These findings raise the possibility that different NSF gene products serve distinct or overlapping functions with the organism.