We identified, in cells transformed by Moloney murine sarcoma virus (M-MuSV clone 124), a protein encoded by the M-MuSV transforming gene, v-mos. An antiserum against a synthetic peptide corresponding to the C terminus of a protein predicted from the v-mos nucleotide sequence specifically recognizes a protein doublet of approximately 37,000 daltons from 35S-methionine-labeled M-MuSV 124-transformed producer cells. By peptide mapping, this protein is almost identical to the 37 kd in vitro translation product from the M-MuSV v-mos gene. Immunoprecipitates from 32P-labeled cells contain a single v-mos-specific phosphoprotein, which has at least six sites of phosphorylation containing phosphoserine. Pulse-chase experiments show that the lower band in the 35S-methionine-labeled doublet is the primary translation product, which is modified, probably by phosphorylation, to yield the upper band. A similar mos protein is immunoprecipitated from HT1-MuSV-transformed cells, but not from uninfected NIH/3T3 cells. These mos proteins are present at very low levels in transformed cell lines. Cells acutely infected with M-MuSV 124, however, transiently contain much higher levels of the mos protein. These high levels coincide with extensive cell mortality.