Previous studies have shown that transforming growth factor beta 1 (TGF-beta 1) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter as effectively as TGF-beta 1. The same c-myc promoter region was required for regulation by both TGF-beta 1 and pRB. These sequences, termed the TGF-beta control element (TCE), lie between positions -86 and -63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-beta 1 treatment of TGF-beta-sensitive but not TGF-beta-insensitive cells. These data indicate that pRB can suppress c-myc transcription and suggest the involvement of cellular factors in addition to pRB in the TGF-beta 1 pathway for the inhibition of c-myc transcription and growth inhibition.