Tumorigenic guinea pig cell lines with mutationally activated N-ras alleles also exhibited up-regulated N-ras mRNA. Mutational activation and mRNA up-regulation were limited to tumorigenic cells; preneoplastic progenitors were unaffected. Therefore, up-regulation occurred at a late stage of carcinogenesis closely associated with acquisition of tumorigenicity. cDNA and S1 protection analysis demonstrated that polyadenylation site of the short N-ras message and the mRNA start sites were different from that reported for human. The promoter region contained no canonical TATA or CCAAT boxes, but exhibited GGGCGG and CCGCCC SPl binding motifs characteristic of growth control genes. Moreover, both mutant and wild-type alleles were up-regulated in a guinea pig line heterozygous for N-ras codon 61. Coordinate N-ras mutational activation and up-regulation in five independent tumorigenic lines with unique chromosome constitutions suggests that both events are required for expression of the neoplastic phenotype.