The influence of insulin and glucose on the glycogen content of isolated trophoblast cells was measured for the first time. The cells were obtained by tryptic digestion of villous tissue from term placentae of 15 healthy women, further purified on a Percoll gradient and enriched by employing a monoclonal anti HLA class-I antibody. The cells stained intensively with PKK1 and exhibited the structural and phenotypical characteristics of intermediate and syncytiotrophoblasts. The mean glycogen content of cells cultured in Dulbecco's Modified Eagle's Medium with 5.5 mM glucose was 17.7 +/- 3.2 micrograms/mg protein, remained constant from days 1-4, and was unaltered by higher glucose concentrations (17 and 28 mM) in the medium. Between 2-5 h after a medium change, the cells contained 50% more glycogen than at the time of replacement. Short term (0-5 h) as well as long term incubations (24 h) with both pathological (10(-8) M) and physiological (10(-9) M) concentrations of insulin had no effect, with respect to either the rate of increase in glycogen or the maximum level. We conclude that the glycogen content of isolated syncytiotrophoblasts in vitro is invariant to extracellular glucose concentrations and is not regulated by insulin. This suggests that cells other than syncytiotrophoblasts and their immediate precursors account for the reported alterations of glycogen content in normal human term placenta in vitro under hyperglycemic or hyperinsulinemic conditions.