Seminolipid (1-O-alkyl-2-O-acyl-beta-3'sulfogalactosyl glycerol) was detected on a micro-scale by high performance liquid chromatography (HPLC) using a UV detector. The benzoylation method of McCluer and Evans ((1973) J. Lipid Res. 14, 611-617) was used to convert seminolipid to a derivative with UV absorption. Total lipid from germinal tissue was benzoylated and the product was separated by Florisil and Sephadex LH-20 column chromatographies. The seminolipid obtained in the effluent from the Sephadex LH-20 column was determined by HPLC. Eighty percent recovery of 35S-labelled seminolipid was obtained by this method. The method was used to determine the seminolipid content of boar spermatozoa: 980 nmoles of seminolipid was found per gram of packed cells.