We prepared an antiserum to a 14-amino-acid-long synthetic peptide derived from the published nucleotide sequence of a Moloney virus-derived MCF recombinant virus. This peptide is not found in the parental Moloney virus gp70 sequence or in other ecotropic viruses so far sequenced. When several tissues of the mouse were examined by Western blot analysis for related molecules, we found that thymocytes from 2-3-mo-old mice, but not splenic T cells or bone marrow cell extracts reacted with this probe. We found that the Mr 70,000 thymic glycoprotein recognized by the anti-peptide antiserum (81-TAg) was present at equivalent levels on peanut agglutinin positive thymocytes in all mouse strains tested. These results indicate that 81-TAg is a differentiation antigen that is related to the substituted portion of an MCF retrovirus.