To investigate the importance of glycolysable substrate for supporting the ability of human sperm to capacitate and penetrate oocytes in vitro, washed spermatozoa were incubated with or without various sugars in BWW culture medium containing pyruvate and lactate. Sperm penetration was assayed using zona-free hamster oocytes. After an 18-h preincubation, glucose (1 mg/ml) supported higher penetration of sperm into oocytes than either mannose or fructose (60.7% vs 28.2% or 21.5%, respectively) at the same concentration. Penetration was even lower when medium contained the nonmetabolizable sugar galactose (2.1% at 1 mg/ml). On the other hand, higher concentrations (5 or 10 mg/ml) of glucose, but not fructose, suppressed penetration, provided the glucose was present throughout the 18-h preincubation. When caffeine, a stimulant of glycolysis in human sperm, was present along with glucose, sperm penetration was enhanced, but only after 6 h of sperm preincubation. The effect was not observed in glucose-free medium, however, where penetration remained low over a 10-h incubation period. In these experiments, the percentage of motile sperm was unaffected by treatment, but the quality of motility was diminished in the absence of glucose. Stimulation of glycolysis may promote capacitation of human spermatozoa in vitro and that optimization of penetrating ability of sperm is dependent upon both the type and concentration of glycolysable sugar present.