The promoter of the Drosophila melanogaster Ras2 gene is bidirectional, regulating an additional gene oriented in the opposite polarity. The two divergently transcribed genes are only 93 bases apart and deletion analysis proved that common cis-acting elements within this promoter region are required for the transcriptional activity of both genes. We cloned the gene paired with Ras2 in the bidirectional promoter and isolated cDNAs corresponding to its mRNA. The Ras opposite (Rop) gene encodes for a 68 x 10(3) M(r) protein which shares sequence homology with the members of a novel Saccharomyces cerevisiae gene family, including the SLY1, SEC1 and VPS33 (SLP1) genes, all of which are involved in vesicle trafficking among yeast cellular compartments. A highly conserved motif in this family is also found in beta-COP, a coat protein isolated from rat Golgi-bound nonclathrin vesicles. Thus, the Rop protein may be a component of one of the vesicle trafficking pathways in Drosophila cells. The Rop gene expression during embryogenesis is restricted to the central nervous system (CNS) and the garland cells, a small group of nephrocytes that takes up waste materials from the haemolymph by endocytosis. Ras2 is also expressed in the embryonic garland cells. In postembryonic stages, the two genes are co-expressed in the larval salivary glands and the central nervous system, and in the adult CNS and reproductive systems. Interestingly, the S. cerevisiae SLY1-20 allele is a suppressor of the loss of the YPT1 gene, a ras-like gene implicated in vesicle translocation, suggesting that the two genes may interact with one another. Since Sec1p and beta-COP may also interact with small GTP-binding proteins of the ras superfamily, it is conceivable that the Rop and Ras2 gene products are not just co-expressed in common tissues, but may also functionally interact with one another in these tissues.