According to experimental studies, 17 beta-estradiol associates with lipoproteins in blood in the form of fatty acid esters. However, concentrations of endogenous estradiol esters in human lipoprotein fractions have not been previously reported. We investigated the distribution of estradiol fatty acid esters between plasma lipoproteins in 10 healthy women during late pregnancy. Following extraction from serum and ultracentrifugally isolated, gel-filtered lipoproteins, estradiol esters were separated from nonesterified estradiol by column chromatography. After saponification and chromatographic purification of the estradiol ester fraction, the concentration of hydrolyzed esters was determined by estradiol time-resolved fluoroimmunoassay. Of total serum estradiol, a mean of 0.7% (549 pmol/liter, n = 10) was in the form of fatty acid esters. Estradiol fatty acid ester concentrations measured in serum and lipoprotein fraction correlated positively (n = 10; r = 0.98; P < 0.001). The majority of lipoprotein estradiol esters, 54%, was recovered in high-density lipoprotein, and 28% in low-density lipoprotein fraction. Most lipoprotein fractions contained undetectable amounts of nonesterified estradiol (< 36 pmol/liter). In conclusion, our results indicate that estradiol fatty acid esters are mostly bound by lipoproteins in blood in vivo.