We evaluated the role of epidermal growth factor (EGF) in the regulation of L–alanine transport in LLC–PK1 renal epithelia. After 2 h of incubation, EGF had no significant effect on L–alanine uptake by LLC–PK1 cells. However, prolonged (16 h) incubation with 2 and 20 ng/ml of EGF resulted in significant increases in sodium–dependent L–alanine uptake as compared with controls. Treatment with 12– O–tetradecanoylphorbol–13–acetate (TPA; 20 ng/ ml) caused a marked increase in sodium–dependent L–alanine uptake after both 2 and 16 h of incubation, and the treatment with TPA (20 ng/ml) EGF (20 ng/ml) for 16 h resulted in significant acceleration of the TPA–stimulated increase in L–alanine uptake by LLC–PK1 cells. Coincubation with H–7 (20 μ M) inhibited both EGF– and TPA–stimulated increases in L–alanine uptake, and genistein (20 μg/ml) blocked the stimulatory effect of EGF in L–alanine transport to the control level. Furthermore, coincubation with cycloheximide (20 μg/ml) for 16 h inhibited both EGF– and TPA–stimulated increases in L–alanine transport to a great extent. The sodium– independent L–alanine uptake was not affected by treatment with either EGF or TPA. These results suggest that the activation of protein kinase C through tyrosine kinase activation plays a role in the EGF effect of stimulating L–alanine transport in LLC–PK1 cells and that the effect is mainly due to increased protein de novo synthesis which occurs after protein kinase C activation.