We have investigated the ability of CHO cells to allow growth of papovaviruses by analyzing viral DNA replication after transfection using the calcium-phosphate co-precipitation technique. These analyses showed that when SV40-containing plasmids were introduced into CHO cells, viral DNA replicated to a level of approximately 1000 copies per T antigen-expressing cell, and neither late proteins nor virus progeny were produced. When polyoma (Py)-containing plasmids were transfected into CHO cells, a ten-fold higher level of Py DNA was present per T antigen-positive cell, and viral capsid proteins and progeny virus were detected, indicating that CHO cells are not equally restricted for all papovaviruses. Infection with intact virions was restricted in both cases. These results indicate that either SV40 or Py DNA introduced into CHO cells are able to express their early viral functions, and that different interactions of cellular proteins involved in the replication machinery with viral nucleic acids and proteins result in different levels of viral DNA synthesis and virus progeny production. We propose that, because of their favorable genetic characteristics, CHO cells should, therefore, provide a valuable experimental system for definition of the cellular contributions to papovavirus replication.