N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase known to have an essential role in intracellular membrane transport events. Recently, cDNA clones encoding a Drosophila melanogaster homolog of this protein, named dNSF, were characterized and found to be expressed in the nervous system. We now report the identification of a second homolog of NSF, called dNSF-2 within this species and report evidence that this ubiquitous and widely utilized fusion protein belongs to a multigene family. The predicted amino acid sequence of dNSF-2 is 84.5% identical to dNSF (hereafter named dNSF-1), 59% identical to NSF from Chinese hamster, and 38.5% identical to the yeast homolog SEC18. The highest similarity was found in a region of dNSF-2 containing one of two ATP-binding sites; this region is most similar to members of a superfamily of ATPases. dNSF-2 is localized to a region between bands 87F12 and 88A3 on chromosome 3, and in situ hybridization techniques revealed expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis.