In A431 cells synchronized by treatment with thymidine, the level of EGF-stimulated tyrosine protein kinase activity in cells in S and G2/M phases was reduced approximately 40% relative to that seen in cells in G1. This decrease in receptor tyrosine protein kinase activity did not correlate with a decrease in cell surface EGF receptor expression, indicating that the reduced activity could not be attributed to receptor loss. EGF-stimulated PI 3-kinase activity was also reduced by approximately 60% during S phase as compared to G1 phase. The change was not due to decreased PI 3-kinase expression since Western blot analyses indicated that cellular p85 levels remained constant throughout the cell cycle. These data suggest that the ability of EGF to stimulate biological responses varies during the cell cycle and implicate cell-cycle-dependent processes in the regulation of EGF-receptor-mediated signaling.