We have determined that there are at least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 in the crude membrane fraction of bovine brain and have purified one of them with a Mr of about 24,000 (24K G) to near homogeneity (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). In this study, we have purified another G protein with a Mr of about 20,000 (20K G) to near homogeneity and have characterized it. 20K G bound maximally about 1.0 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein, with a Kd value of about 50 nM. [35S]GTP gamma S binding to 20K G was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP; it was also inhibited by pretreatment with N-ethylmaleimide. 20K G hydrolyzed GTP to liberate Pi, with a turnover number of about 0.01 min-1, and was not copurified with the beta gamma subunits of the regulatory G proteins of adenylate cyclase. 20K G was not recognized by the antibody against the ADP-ribosylation factor for the stimulatory regulatory G protein of adenylate cyclase. Peptide map analysis showed that 20K G was not a proteolytic product of 24K G. The partial amino acid sequence of 20K G was almost identical with that deduced from the rho gene. The amino acid composition of 20K G was similar to that of the rho gene product. These results suggest that 20K G is the rho gene product and that this G protein is present in bovine brain membranes.