We examined the effects of elastase [EC 3.4.21.11] on lipogenesis, antilipolysis, and pyruvate dehydrogenase activity in rat epididymal adipose tissue in comparison with those of insulin and trypsin [EC 3.4.21.4]. The rate of conversion of [3-3H]-glucose into lipid in fat cells was stimulated by elastase, trypsin, and insulin. When fat pads were incubated with elastase, trypsin, or insulin in the presence of glucose, pyruvate dehydrogenase activity in the homogenate of the incubated fat pads was markedly increased. In the absence of glucose, elastase did not increase pyruvate dehydrogenase activity, though trypsin and insulin showed a slight but significant increase. Further, the increasing effect of elastase in the presence of glucose was inhibited by the addition of 3-O-methylglucose or phlorizin to the incubation mixture of the fat pads. Trypsin and insulin still showed a significant increase under similar conditions. When the homogenate of intact fat pads was incubated with elastase, the pyruvate dehydrogenase activity was progressively decreased with increase in the concentration of elastase. Concanavalin A showed an additive effect on the pyruvate dehydrogenase activity increase caused by elastase, whereas such an effect was not observed with insulin or H2O2. The stimulation of lipolysis by epinephrine in the fat cells was not suppressed by elastase, in contrast to trypsin and insulin. These results suggest that elastase reacts with the cell surface, facilitates glucose transport into the fat cells, and consequently affects glucose and lipid metabolism by somewhat different mechanisms from those of insulin and trypsin.