The present report describes in vitro experiments with golden hamster sperm designed to determine whether there is any relationship between sperm phospholipid methylation and capacitation and/or the acrosome reaction. Washed cauda epididymal hamster sperm were incubated in a capacitation medium containing [methyl-3H] methionine. After 0.5, 1.5, 2.5 and 3.5 h of incubation, sperm were extracted with a chloroform:methanol:2 N HCl mixture to extract total phospholipids. Liquid scintillation counting revealed that the methyl-3H-group was incorporated into phospholipids with maximum incorporation at 3.5 h and an increase of 50% between 2.5 and 3.5 h. Uptake of labeled methionine by sperm reached its plateau by 1.5 h of incubation. Some sperm were capacitated by 3.5 h because that is the time at which the rate of acrosome reactions began to increase and because at least 50% of them were able to undergo the acrosome reaction 10 min after the addition of the fusogen lysophophatidylcholine (LPC) at 3.5 h but not at 2.5 h. Homocysteine thiolactone and 3-deazadenosine, inhibitors of transmethylation, inhibited incorporation of methyl-3H into phospholipids at 3.5 h by approximately 90% and also inhibited LPC-induced acrosome reactions by 60%. Separation of methylated sperm phospholipid by thin-layer chromatography demonstrated the presence of 3H-labeled phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and to a lesser extent phosphatidylcholine. In addition, an unidentified lipid was also highly labeled. These results strongly suggest a positive correlation between phospholipid methylation and capacitation and/or the acrosome reaction of hamster sperm in vitro. Possible mechanisms for phospholipid methylation involvement in these events are discussed.