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Publisher Summary Seminolipid 2-O-acyl-1-O-alkyl-3-O-β-D-(galactosyl 3-sulfate)-glycerol was a major glycolipid in boar testes and spermatozoa and was present in higher content in spermatozoa than in testes. This chapter discusses the determination by high-performance liquid chromatography (HPLC) of the decrease of seminolipid content in rats with vitamin A deficiency. It describes various tests to be performed on rats and mouse. Seminolipid 2-O-acyl-1-O-alkyl-3-O-β-D-(galactosyl-3-sulfate)-glycerol was a major glycolipid in boar testes and spermatozoa. The chapter presents the findings of an investigation that suggest that seminolipid biosynthesis is closely related to spermatogenesis and change of seminolipid content in vitamin A deficient rat test. A new method for the micro scale determination of seminolipid by HPLC. Total lipids were extracted from rat testes and benzoylated with pyridine and benzoyl chloride. The benzoylated derivatives were applied to a Florisil column and eluted with chloroform, chloroform-acetone, and chloroform methanol. The fraction eluted with chloroform-acetone was evaporated to dryness, aliquots of the chloroform solution of each residue was analyzed by H.p.l.c. on a packed μBondapak C18 column with 90% methanol containing 0.01% tetrabutylammonium hydroxide as an eluting solvent.