1-O-Alkyl-2-O-acyl-3-O-beta-D-(3'-sulfatoxygalactopyranosyl)-sn- glcerol (SGG) and its nonsulfated analog (GG) are the major glycolipids of rat testis. Aspects of the biosynthesis and metabolism of these two lipids have been investigated by determining their specific activities at various times after injection of [16-14C]palmitic acid, [1-14C]cetyl alcohol, and D-[1-14C]galactose into the testes of the adult rats. Evidence was obtained from studies with each of these three radioactive compounds that is consistent with the interpretation that GG exhibits a precursor relationship to SGG in vivo. The turnover time of GG, as estimated from the use of each of the three precursors, ranged between 21 and 69 h. In contrast, a slow increase of radioactivity in SGG was observed following injection of each of the three precursors, a plateau value being reached between 72 and 168 h. The radioactivity in the acyl, alkyl, and galactosyl moieties of SGG thereafter remained quite constant for another 21 days. Small amounts of monoalkylmonoacylglycerol were detected in rat testis. Radioactive studies indicated that this compound could be a precursor of GG and (or) monoalkyldiacylglycerol, another lipid that was also detected in rat testis. The results are consistent with the concept that the synthesis of SGG occurs primarily at an early stage of spermatogenesis and that the various moieties of this lipid exhibit almost complete metabolic stability during the subsequent complex stages of this process.