Rifamycin SV and several of its derivatives were examined for their ability to inhibit the replication of Gross murine leukemia virus in intact Swiss mouse embryo cells growing in culture. Antiviral activity was observed with rifamycin SV, 3-formyl rifamycin SV: dipropylhydrazone and benzyl dimethyl rifampicin, when the compounds were tested against this RNA tumor virus by means of the UV-XC plaque-reduction procedure. However, several of the rifamycin derivatives which strongly inhibited the viral RNA-directed DNA polymerase reaction in cell-free systems failed to inhibit virus replication in intact cells at noncytotoxic concentrations.