To examine whether pre-β-high-density lipoprotein (HDL) may be involved in regulation of human placental lactogen (hPL) release, pre-β-HDL was isolated from term pregnancy serum, and the effect of purified pre-β-HDL on hPL release from trophoblast cells was examined after 1 h of exposure. Pre-β-HDL stimulated a dose-dependent increase in hPL release with half-maximal stimulation at a dose of 300–400 μg/ml, which is within the normal physiological range during pregnancy. Analysis of pre-β-HDL and α-HDL in serum from pregnant women at different stages of gestation (determined by Western blot analysis) indicated that the pre-β-HDL-to-α-HDL ratio increased linearly after the 10th week of gestation (r = 0.88,P < 0.001), reaching a maximum sixfold greater than that of nonpregnant women. The increase in serum pre-β-HDL during pregnancy paralleled that of plasma hPL concentrations (r = 0.93,P < 0.001). Two-dimensional electrophoresis indicated that the increase in pre-β-HDL was due primarily to an increase in pre-β₁-HDL and pre-β₂-HDL, two of the three forms of pre-β-HDL present in blood. These results suggest a role for pre-β-HDL in the regulation of hPL expression during pregnancy.