Experiments have been conducted to examine the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on growth-related signaling in vascular smooth muscle cells (SMCs). A 40% reduction of peak DNA synthesis was observed in SMCs only when TCDD was added during the G0/G1 transition of the cell cycle. Enhanced phosphorylation of several endogenous proteins during this period was coincident with increased tyrosine kinase activity as early as 15 min following TCDD challenge. No changes in protein phosphorylation status occurred in cells treated with TCDD during the G1/S transition or during S phase. Cotreatment of quiescent SMCs with 10 nM TCDD and serum for 3 h reduced serum-inducible binding activity to a 12-O-tetradecanoyl phorbol 13-acetate responsive element (TRE) by approximately 40%. No alterations of constitutive TRE binding were observed in quiescent SMCs treated with TCDD for up to 5 h. These data show that mitogen-related signaling in vascular SMCs is modulated by TCDD selectively during the G0/G1 transition, and these effects influence the growth behavior of these cells.