Potential mechanisms by which transforming growth factor beta 1 (TGF beta 1) inhibits cell growth include suppression of transcription of genes required for proliferation and inactivation of enzymatic activities that regulate progression through G1. To clarify which events are important for TGF beta 1 signaling, we have defined more precisely the times in G1 when the mouse keratinocyte cell line BALB/MK is sensitive to TGF beta 1-induced inhibition. TGF beta 1 is capable of inhibiting BALB/MK cell growth when the inhibitory factor is added to cells at any point before the G1-S transition. Synchronization at the G1-S boundary with mimosine, an inhibitor of initiation of origins of DNA replication, followed by release into S phase in the presence of TGF beta 1, indicated that cells lose sensitivity to TGF beta 1 as they enter the replicative phase. It is interesting that TGF beta 1 can inhibit BALB/MK cell growth late in G1, at a time when cell cycle progression is not blocked by an inhibitor of RNA polymerase II-mediated transcription. 5,6-dichlorobenzimidazole riboside (DRB). In addition, the ability of TGF beta 1 to inhibit entry into S phase in late G1 is unaffected by concurrent treatment with DRB. The data suggest that, at least when added to cultures that are in the latter few hours of G1, TGF beta 1 can inhibit cell cycle progression through mechanisms that do not involve stimulation or inhibition of gene expression at the transcriptional level.