Cultured human pancreatic cancer cells produce transforming growth factor-alpha (TGF-alpha), a potent mitogenic polypeptide. In the present study, we investigated the regulation of TGF-alpha mRNA expression in T3M4 human pancreatic carcinoma cells. TGF-alpha mRNA levels were quantitated by densitometric analysis of autoradiographs obtained following hybridization of size-fractionated cytoplasmic RNA with 32P-labeled cRNA coding for human TGF-alpha. There was a twofold increase in TGF-alpha mRNA levels at 2 h following addition of either epidermal growth factor (EGF) or TGF-alpha. However, TGF-alpha mRNA levels declined to near basal levels by 10 h. At 2 h, one-half maximal stimulation of TGF-alpha mRNA levels occurred at 1 nM and maximal stimulation at 4 nM of either EGF or TGF-alpha. The transcriptional inhibitor actinomycin D (Act D) and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), mimicked the actions of EGF and TGF-alpha. These findings indicate that the regulation of TGF-alpha mRNA expression in T3M4 cells is complex, and is mediated, in part, via the EGF receptor.