Verots S3 cells derived from the African green monkey kidney were revealed to contain nine types of sulfoglycolipids by incorporating [35S]sulfate. These sulfated glycolipids were separated by DEAE-Sephadex column chromatography and preparative thin-layer chromatography (TLC). The major sulfoglycolipids were characterized using TLC, gas–liquid chromatography (GLC), mass spectrometry, solvolysis, TLC immunostaining, and nuclear magnetic resonance spectra as follows: V1, SM4s (GalCer I3-sulfate); V2, SM3 (LacCer II3-sulfate); V3, SM2a (Gg3Cer II3-sulfate); V4, globopentaosyl ceramide sulfate (Gb5Cer V3-sulfate); V5, (Gg4Cer II3-sulfate, IV3-NeuAc); V6, SB1a (Gg4Cer II3, IV3-bis-sulfate); and V8, (Gg4Cer II3-NeuAc, IV3-sulfate). Both V5 and V8 were sulfated gangliosides comprising both N-acetyl neuraminic acid and sulfate, and this was the first report on V8. A minor component V7 was identified as SM1a (Gg4Cer II3-sulfate) based on its behavior in TLC, GLC, and liquid secondary ion mass spectroscopy. It was postulated that this substance was a precursor of V6 (SB1a) and V5 (Gg4Cer II3-sulfate, IV3-NeuAc), and to date, its presence has not been demonstrated in nature. Another minor component V9 was identified as glucosyl ceramide sulfate based on its migration in TLC and GLC. This renal cell line was shown to be an excellent model for studying the metabolism and function of sulfoglycolipids.