Monoclonal antibodies have been generated against disrupted mammary tumor viruses isolated from Mus musculus, Mus cervicolor, and Mus cookii. Monoclonal antibodies directed against the M.W. 36,000 external glycoproteins of these viruses demonstrate the presence of multiple epitopes, i.e., distinct antigenic determinants, within these glycoproteins. These epitopes represent type, group, and interspecies determinants. Monoclonal antibodies have also been used to define multiple epitopes on the M.W. 28,000 major internal polypeptides of murine mammary tumor viruses that exhibit both type- and group-specific determinants. The monoclonal antibodies to the M.W. 36,000 glycoprotein and the M.W. 28,000 polypeptide have been used to distinguish all six mammary tumor virus isolates of M. musculus from each other, including both endogenous and exogenous viruses from the same strain, and a new virus isolate from BALB/c mice. With the use of the immunoperoxidase technique, the monoclonal antibodies generated have been used to demonstrate a heterogeneity of expression of mammary tumor virus gene products in primary mammary tumors of three Mus species. These studies have revealed that a given antigenic determinant may be expressed differentially in mammary tumors of two different Mus species, among mammary tumors of the same Mus species, and, at times, in different areas of the same mammary tumor.