We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.