We have shown previously that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) under cell-free conditions causes a significant rise in protein tyrosine kinase activity of cytosol from male guinea pig adipose tissue, and that such an effect of TCDD is Ah-receptor dependent. In the present study, we obtained evidence indicating that c-Src protein kinase is the protein kinase activated by TCDD and that this kinase is associated specifically with the Ah-receptor-complex proteins in cytosol from adipose tissue and liver of guinea pig and liver of C57BI/6J mouse, and in NIH 3T3 mouse fibroblast cells. Here, we present evidence that c-Src protein is functionally attached to the Ah-receptor (AhR) and is specifically activated upon ligand binding. This conclusion is based on several lines of evidence: (a) TCDD caused activation of protein tyrosine kinase activity when administered directly to purified Ah-receptor immunoprecipitate; (b) this stimulatory effect of TCDD was abolished when the cytosol was immunodepleted of c-Src protein or Ah-receptor protein by preincubating with anti-c-Src or anti-Ah-receptor antibody, followed by the addition of TCDD to the remaining portions of cytosol; (c) when Ah-receptor immunoprecipitate was incubated with TCDD, and the kinase(s) released to the supernatant was analyzed on autoradiography of two-dimensional (2D) electrophoresis, 32P-labeled c-Src protein was recognized; (d) the same 32P-labeled-phosphoprotein with M(r) = 60 kDa and pl = 6.1 was found in the immunoprecipitate with anti-c-Src antibody on 2D autoradiograms; (e) this same phosphoprotein disappeared when the supernatant of the Ah-receptor immunoprecipitate was immunodepleted of c-Src protein by anti-c-Src antibody; and (f) a structure-activity relationship study with TCDD and three dioxin-congeners revealed a rank order for their potency in activation of c-Src kinase activity to be identical to that of previously determined toxicity indices: i.e., TCDD > 1,2,3,7,8-pentachlorodibenzo-p-dioxin (1,2,3,7,8-PCDD) > 1,2,4,7,8-pentachlorodibenzo-p-dioxin (1,2,4,7,8-PCDD) > 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). Consistent with these results, TCDD-induced c-Src kinase activity was abolished when c-Src immunoprecipitate's suspension was preincubated with 0.1 or 1 microM alpha-naphthoflavone (AhR blocker) for 10 min prior to the addition of TCDD. In addition, pretreatment of 3T3 fibroblast cells with 3-methylcholanthrene abolished TCDD-induced c-Src kinase activity in AhR-immunoprecipitate. We conclude that c-Src protein kinase is associated specifically with the AhR complex along with hsp90 in the cytosol of these cells and that upon ligand binding to the Ah-receptor subunit, c-Src is activated and released from the complex.