Polyoma wild-type virus replicates in most murine differentiated cells but fails to produce virus in murine embryonal carcinoma cells. Polyoma host range mutants have been isolated that replicate in several nullipotential embryonal carcinoma cell lines but fail to replicate in a pluripotential cell line. The final virus yield of these host range mutants is dependent upon the multiplicity of infection in both differentiated cells and nullipotent embryonal carcinoma cells. Two independently derived host range mutants contain the same single base pair change (A.T to G.C) and a 33- and 67-base pair duplication of those viral DNA sequences containing this point mutation. This duplication of viral DNA is located at 69 map units on the polyoma genome on the late gene side of the origin of viral DNA replication (70.5 map units). This type of mutation suggests several models to explain the polyoma host range restriction in embryonal carcinoma cells.