Two-dimensional gel electrophoretic analysis (NEPHGE, IEF) of the [32P]-orthophosphate-labeled proteins synthesized throughout the cell cycle of transformed human amnion cells (AMA) revealed two phosphoproteins (dividin, Mr = 54,000, pl = 8.4; IEF 59dl, Mr = 27,000, pl = 5.7) that are present mainly in S-phase cells. These proteins are first detected at the end of G1, near the G1/S transition border, and their levels reach a maximum late in S-phase. Together with the previously identified nuclear protein cyclin, these phosphoproteins are likely candidates for proteins that may play a role in the regulation of the onset of DNA synthesis and cell division.