Mouse embryonal carcinoma cells are refractory to infection by wild-type polyoma virus, the infection process apparently being blocked at a stage after adsorption and penetration but before early protein synthesis. Polyoma virus mutants capable of productive infection of mouse embryonal carcinoma cells have been isolated and these mutants all have DNA sequence alterations in a noncoding region near the origin of replication of the viral genome. PyF101 and PyF441 are two mutants selected for their ability to infect the embryonal carcinoma cell line F9. Here we show that these PyF mutants do not rescue replication of wild-type polyoma during a mixed infection of F9 cells. The mutant and wild-type DNAs were distinguished on the basis of restriction fragments obtained by digestion with Msp I or BstNI, and no wild-type DNA was detected in F9 cells coinfected with wild-type polyoma and with either PyF101 or PyF441. The mutant viruses do not appear to inhibit wild-type replication during a mixed infection because both mutant and wild-type DNAs can replicate efficiently in coinfected 3T6 cells which are permissive for both mutant and wild-type viruses. A double mutant having the PyF101 mutation and the ts-25E temperature-sensitive mutation in polyoma large tumor antigen was constructed and found to be temperature-sensitive for replication in F9 cells. This double mutant, designated PyFts-1, can be rescued in F9 cells at the restrictive temperature by coinfection with PyF441. These results suggest that the PyF mutations affect two processes in F9 cells, one involving expression of polyoma early genes and a second involving viral DNA replication.