The viral mos gene encodes a cytoplasmic transforming protein termed p37mos. Evidence gathered from a number of experimental approaches is consistent with p37mos having a serine/threonine protein kinase activity. To gain further understanding of the p37mos-associated biochemical activity, we constructed a mutation in the v-mos gene by oligonucleotide-directed mutagenesis yielding a histidine to tyrosine substitution at residue 221 in p37mos. Based upon nucleotide sequences, the histidine residue at the corresponding position is conserved in all the serine/threonine protein kinases from yeast to man, and is absent in protein-tyrosine kinases. The mutant p37mos (Tyr-221) was expressed in yeast and assayed for kinase activity. The mutant protein was inactive as judged by a loss of autophosphorylation activity in vitro, thus providing further support for the conclusion that p37mos is a protein kinase. When the mutant v-mos gene was introduced into a retroviral vector, pDD102, and assayed for focus-forming ability on NIH/3T3 cells, it was found to be inactive at both 37 and 30 degrees. In contrast, the wild-type v-mos had transforming activity at both temperatures. These results extend our earlier findings on the correlation between transforming ability and protein kinase activity. A histidine to tyrosine substitution at the corresponding position of the v-mos protein and the yeast CDC28 gene product causes a similar effect on the kinase activity. Therefore, this residue and/or the sequence near the N-terminal side of the conserved predicted phosphate transfer domain, near the middle of the complete catalytic domain, might be specifically involved in the catalytic activity of serine/threonine protein kinases in general.