Exposure of cultured glomeruli to benzo[a]pyrene (BaP), a carcinogenic hydrocarbon, modulates mesangial and visceral epithelial cell proliferation in vivo and in vitro. The present studies were conducted to characterize mitogenic signaling profiles of cultured glomeruli following repeated cycles of BaP challenge. Enhanced rates of DNA synthesis were observed by the third passage in randomly cycling cultures after single or repeated carcinogen exposure. This response was characterized by upregulation of mitogenic sensitivity during early cell cycle transit, and increased cell numbers under restrictive growth conditions. The mitogenic response to platelet-derived growth factor (0.5 to 25 ng/mL), acidic fibroblast growth factor (2.5 to 10 ng/mL), basic fibroblast growth factor (0.05 to 5 ng/mL), epidermal growth factor (0.5 to 5 ng/mL), or conditioned medium was not enhanced by hydrocarbon challenge. BaP-treated cultures exhibited anchorage-independent growth and increased expression of hepatocyte growth factor mRNA and E-cadherin protein. Binding of activator protein-1 to DNA was enhanced in BaP-treated cells, but this change did not involve truncation or mutation of the c-jun delta region. Collectively, the data demonstrate that repeated cycles of BaP injury alter mitogenic signaling profiles in cultured glomerular cells. These alterations may contribute to deregulation of proliferative control following carcinogen exposure in vivo.