Using an expression cloning assay, we have isolated a novel cDNA, referred to as rsp-1, which suppresses the v-Ras-transformed phenotype. When introduced into NIH 3T3 fibroblasts under the control of a metallothionein promoter, rsp-1 confers resistance to v-Ras, but not to v-Mos or v-Src, and inhibits growth of the cells. The rsp-1 cDNA contains a 831-bp open reading frame encoding a 277-amino-acid leucine-rich protein. The rsp-1 cDNA exhibits no significant homology to sequences in the DNA data bases. However, searches of the protein data bases revealed that it contains a series of leucine-based repeats which are homologous to the leucine repeats found in the regulatory region of the yeast adenylyl cyclase, rsp-1 specific RNA is detectable in a wide variety of cell lines and tissues, and the gene is conserved among eukaryotic species. These data suggest that rsp-1 plays a role in Ras signal transduction.