Nonproducer cells of STU mouse origin (Sac) transformed with Moloney murine sarcoma virus (Mo-MSV) and superinfected with the amphotropic murine C-type virus strain AP 129 unexpectedly formed polykaryocytes. The multinuclear giant cells appeared about 30 days after AP 129 infection as confirmed in three independently performed experiments. The majority of the Sac-cells became involved in syncytium formation. The polykaryocytes disappeared during continued culture transfers. Various cell lines obtained by limiting dilution showed the same reactivity as the parental cell line. Release of sarcoma-helper virus complex was observed before and during the appearance of the polykaryocytes as well as after their disappearance at about day 50 after infection. In normal STU mouse embryo fibroblasts, normal Balb/3T3 cells or another Mo-MSV transformed nonproducer cells (MSV85 C13; Balb origin) infected with AP 129 no polykaryocytes developed. The sarcoma-helper virus complex released from AP 129 infected Sac-cells led to transformation of cultured cells from different mammalian species (mink, goat, dog). However, when observed for at least 30 days these cultures did not show polykaryocyte formation.