Protein kinase C (PKC) activation stimulates transport system X AG ؊ for anionic amino acids in cultured human fibroblasts (Franchi-Gazzola, R., Visigalli, R., Bussolati, O., and Gazzola, G. C. (1994) FEBS Lett. 352, 109 -112). To identify which PKC isoform is responsible for this effect, aspartate transport through system X AG ؊ , PKC activity, and the subcellular distribution of PKC isoforms have been studied before and after treatment with phorbol 12,13-dibutyrate (PDBu) in fibroblasts maintained at low serum for 1 (control cells) or 7 days (quiescent cells). In control cells aspartate transport and PKC activity in the particulate fraction were stimulated by short term PDBu treatment; both stimulatory effects were down-regulated by a prolonged exposure to the phorbol. In contrast, in quiescent cells aspartate transport and particulate PKC activity were higher than control under basal conditions, unaffected by a short term PDBu treatment, and lowered by a prolonged incubation with the phorbol. In both control and quiescent cells a short term PDBu treatment modified PKC␣ distribution, increasing its membrane-associated fraction. PKC␦ was mostly in the soluble fraction and scarcely sensitive to PDBu. A brief exposure to PDBu increased membrane-associated PKC⑀ in control but not in quiescent cells. In these cells ⑀ isoform was found exclusively in the particulate fraction even in PDBuuntreated cells. A prolonged PDBu treatment caused a partial down-regulation of membrane-associated PKC⑀ in control cells and its marked decrease in quiescent cells. It is concluded that PKC-dependent changes in system X AG ؊ activity parallel the behavior of PKC⑀, thus suggesting a specific role for this isoform in system X AG ؊ regulation.