We have previously isolated mouse mammary tumor virus (MMTV) host range variants by serial virus passage in feline cells. These variants productively infect cells of a broad range of species but replicate most efficiently in feline cells. We report here the isolation of a series of novel MMTV host range variants that have the ability to replicate with high efficiency in murine, rat, canine and human cells, respectively; these variants were isolated by serial virus passage in cells of each respective species. These new variants, furthermore, all retained their ability to efficiently replicate in feline cells, and each exhibited unique host range properties. The novel MMTV variants obtained from murine, rat, feline, canine, and human cells showed no overt evidence of recombination with endogenous type-C viruses in that they retained their antigenic reactivities in group-specific radioimmunoassays for MMTV polypeptides, and were unreactive for type-C virus proteins when tested by radioimmunoassays and DNA polymerase assays. These novel MMTV host range variants now broaden the spectrum of studies that can be undertaken involving MMTV replication and the initiation and promotion of virus-mediated mammary cell transformation.