The rate at which [14C] phosphatidylcholine became associated with rat caput, corpus, and cauda epididymal sperm in vitro was determined. Within 30 sec all sperm types bound significant amounts of [14C] phosphatidylcholine and maximum levels were reached within 8 min. However, throughout the remainder of the 32 min incubation period, there was a gradual decline in the amount of [14C] phosphatidylcholine which remained associated with sperm. Regional differences in the affinity of epididymal sperm for phosphatidylcholine also were demonstrated. Caput sperm had the greatest affinity for [14C] phosphatidylcholine followed by corpus and cauda sperm. After 2 h incubation, significant amounts of [14C] glycerylphosphorylcholine ([14C] GPC) were observed in caput, corpus, and cauda sperm and their corresponding incubation media, suggesting that epididymal sperm have the ability to convert [14C] phosphatidylcholine to [14C] GPC. The gradual decline of [14C] phosphatidylcholine associated with sperm between 8 and 32 min incubation is consistent with the synthesis of [14C] GPC from [14C] phosphatidylcholine. Principal cells, isolated from dispersed caput epididymal tissue by centrifugal elutriation, synthesized and secreted significant amounts of [14C] GPC when incubated with free [14C] phosphatidylcholine or [14C] phosphatidylcholine-labeled sperm. The total [14C] GPC produced from the co-incubation of principal cells with [14C] phosphatidylcholine-labeled sperm was comparable to the total predicted, using the rates of GPC synthesis obtained when the two cell types were incubated alone. When sperm or principal cells were incubated alone, an average of 58% of the GPC synthesized was secreted into the incubation medium. However, 71% of the GPC produced was secreted into the medium when the two cell types were incubated together, suggesting that GPC secretion by one or both cell types was stimulated. It was concluded that high GPC levels in the epididymal fluid are sustained by sperm and principal cells which synthesize and secrete GPC by degrading lecithin that is lost from sperm during epididymal transit. The observation that buffer that contains bovine serum albumin (BSA) converts [14C] phosphatidylcholmne to [14C] GPC suggested that BSA contains phospholipase A and lysophospholipase.