We have introduced a genomic DNA clone of a mutated human N-ras gene from a T-cell leukemia cell line into a retroviral vector equipped with a neo resistance gene and with SV40 and pBR322 origins of replication. The helper free N-ras virus, which was recovered after transfection of the construction in the psi 2 packaging cell line, contained a correctly spliced N-ras gene. Proviral DNA was amplified in cos cells and subsequently cloned in bacteria. Nucleic acid sequence analysis of the activated N-ras gene revealed a point mutation at codon 12 resulting in a glycine to aspartic acid substitution. The N-ras virus was able to transform mouse fibroblastic cell lines, but failed to fully transform mouse primary embryo fibroblasts. MoMuLV or amphotropic 4070A pseudotypes of the virus were injected intraperitoneally into newborn mice. The MoMuLV pseudotype produced only helper-virus-induced leukemias. The amphotropic pseudotype caused fibrosarcomas after a long latent period. The results of these and other in vivo experiments are discussed in relation to known pathogenic effects of other retroviruses carrying H-ras or K-ras genes.