Prostaglandin (PG) E2 binding to fat cells and its consequent antilipolytic effect have been studied in experiments using laboratory animals, but no binding studies have yet been reported using adipocytes from humans. Consequently, we have characterized PGE2 binding to human isolated fat cells to compare the apparent binding constant to the IC50 for the antilipolytic effect of PGE2. Our data indicate that human fat cells contain binding sites that specifically recognize prostaglandins of the E series and demonstrate stereospecific recognition of the more potent of two 15-methyl-PGE2 analogues. There was no evidence for rapid metabolism of PGE2 by isolated adipocytes such as occurs in lung and liver tissue. A double-reciprocal plot of binding data obtained at saturation using [3H]PGE2 and increasing concentrations of PGE2 indicated a single class of binding sites with an apparent binding constant (0.54 nM) that agreed well with the IC50 (0.26 nM) for the antilipolytic effect of PGE2 we observed in human fat cells. The findings from these binding and lipolysis studies are in general agreement with published observations using adipocytes from rodents and provide evidence that the conclusions reached from previous studies of laboratory animals are relevant to adipocyte physiology in humans.