The lipid class compositions of freshly isolated and overnight maintained human sebaceous glands were determined using high performance thin layer chromatography and were found to be broadly similar. The lipid classes identified in freshly isolated glands were broadly similar to those generated from various radiolabeled precursors in vitro, although lower amounts of wax/sterol esters and cholesterol were observed in vitro. We determined the glycogen content of sebaceous glands and showed that during their incubation with several radiolabeled substrates, with the exception of [U-14C]glucose, significant glycogen breakdown occurred, thus providing acetyl-CoA and NADPH that could affect the pattern of lipids synthesized. We examined glycogen-depleted glands and found that their rates and pattern of lipogenesis resemble that of nondepleted glands, except that the squalene/triacylglycerides ratio for acetate and glutamine doubled from 1.6:1 to 3.4:1 and from 0.7:1 to 1.4:1, respectively. We have shown that exogenous glycerol reduces the squalene/triacylglycerides ratio from acetate from 3.4:1 to 1.7:1, suggesting that glycogen-derived glycerophosphate is important in triacylglycerides synthesis from acetate. Moreover, glands express a glycerokinase activity that may fully account for the rate of triacylglycerides synthesis seen in vitro. We conclude that glycerophosphate generation may be as important as NADPH generation in explaining the directing effects of different substrates within the sebaceous gland.