A cell line (SMKT-R3) established from renal cell carcinoma was characterized by sulfolipids and glycolipid sulfotransferases. It was found by analyzing glycolipids extracted from SMKT-R3 cells that sulfolipids constituted a large part of acidic glycolipids. When SMKT-R3 cells were metabolically labeled with sodium [35S]sulfate, the incorporation of the radioactivity was detected in accordance with SM4, SM3 and SM2, which were major sulpholipids found in the cells, by autoradiography of thin layer chromatogram of the acidic glycolipid extracts from the cells. Markedly high activity level of glycolipid sulfotransferases toward GalCer and LacCer as substrates was observed in SMKT-R3 cells. Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) and a protein kinase C inhibitor on glycolipid sulfotransferases were investigated in SMKT-R3 cells. The treatment with TPA caused a dose- and a time-dependent reduction of the enzymes activities. Similarly, H-7 and staurosporine, which are inhibitors of protein kinase C, reduced the glycolipid sulfotransferase activities. These results indicate that the glycolipid sulfotransferase activities are mediated by protein kinase C in SMKT-R3 cells. On the other hand, the treatment of SMKT-R3 cells with epidermal growth factor (EGF) was associated with the increase of the glycolipid sulfotransferase activities in a dose-dependent manner. However, this effect of EGF was counteracted by the pretreatment with TPA or H-7. These findings suggest that EGF induces the glycolipid sulfotransferase activities through protein kinase C.