(3)H-labeled complementary DNA(sarc), complementary to the murine sarcoma virus (MSV)-specific portion of the Moloney MSV (M-MSV) genome, was prepared. M-MSV-specific RNA was then quantitated in the cytoplasm of several M-MSV-transformed, non-virus-producing, clonal NIH 3T3 cell lines. These lines, designated 71 N clones 5, 6, and 3, have been characterized previously by the degree to which they exhibit transformation properties and transcribe Moloney murine leukemia virus-related RNA (S. Salzberg and M. Green, J. Virol. 13:1001-1004, 1974; N. Tsuchida and M. Green, J. Virol. 14:587-591, 1974). By the criteria of cell morphology and agglutination by concanavalin A, cells of clone 5 are highly transformed, cells of clone 6 are almost normal in the sense that they resemble the parent NIH 3T3 cells, and cells of clone 3 are phenotypically intermediate. In the present study, the amounts of cytoplasmic MSV-specific RNA correlated well with the relative degrees of transformation of the cell lines, varying over 35-fold between the least transformed (clone 6) and most transformed (clone 5) lines. Superinfection of either clone 5 or clone 6 with Moloney murine leukemia virus resulted in a fivefold increase in the MSV-specific RNA in the cell cytoplasm. Evidence from (3)H-labeled complementary DNA:cell DNA hybridization studies indicated that the quantity of M-MSV-specific RNA in the nonproducer lines was not directly related to DNA provirus copy number in the cell DNA. Although clones 5 and 6 differ greatly in transformation characteristics and in MSV-specific RNA content, they each apparently contain about two copies of MSV-specific DNA sequence per haploid genome. Thus, factors such as site of provirus integration may be of primary importance in determining virus-specific transcription and cell transformation.