Seven clonal lines of Fischer rat cells transformed with ts-a mutants of polyoma virus were studied. Four clones are characterized by a temperature-sensitive (ts) and three clones by a temperature-insensitive-transformed phenotype. Six clones have retained a functional though temperature-sensitive large T antigen, as judged by a 10- to 20-fold amplification of viral sequences in clones grown at low temperature compared to those grown at high temperature. No amplification is observed in one non-ts clone. As analyzed by Southern blotting, no obvious difference appears in the integration pattern of viral sequences in ts and non-ts clones concerning the number of sites of genome integration, the presence or absence of tandem repeats of the viral genome, or the absence of specific viral sequences. In autoradiograms of gel-electrophoresed immunoprecipitates, no correlation can be drawn between the amounts of either large T antigen or middle T antigen and the type of transformed state of the clones under the conditions tested. In assays of the middle T-antigen-associated kinase, no reproducible difference can be observed between the non-ts and ts clones. Finally, no correlation was observed between a temperature-insensitive phenotype and the production of an N-terminal fragment of large T antigen. Thus the molecular basis for the difference between ts-a transformants with ts or non-ts phenotypes remains elusive.