Maternal serum pools obtained from healthy women throughout normal pregnancy were fractionated by a combined immunoaffinity chromatography, preparative PAGE, and electroelution procedure. Inhibin A and the pro-αC region of the inhibin α-subunit were determined in the eluted fractions by specific ELISAs, and the profiles of immunoactivity characterized in terms of molecular weight and percent recovery. The molecular weight patterns of inhibin A and pro-αC in serum during early pregnancy (<19 wk gestation) showed peaks between 25-40K and approximately 60K, consistent with the presence of known mature and larger precursor inhibin forms. However, during late pregnancy (>19 wk gestation), an increase in the proportion of smaller molecular weight forms (from 2% to ∼25%) of inhibin A and pro-αC of unknown structure were observed in the less than 30K and less than 25K regions, respectively. To assess whether this change in molecular weight distribution in late pregnancy was related to the method of serum collection, serum and plasma from women during early and late pregnancy were collected and snap-frozen. Three pools [one from early pregnancy (12-15 wk), two from late pregnancy (28-39 wk)] of serum and plasma were then fractionated as described above. No differences in molecular weight patterns of inhibin A and pro-αC were observed between serum and plasma pools obtained in early pregnancy. However, in late pregnancy there was a reduction in the proportion of low molecular weight forms between serum (25% inhibin A, 35% pro-αC) and plasma (12% and 17%, respectively), but not to the low levels seen in early pregnancy. Incubation of iodinated 30K human inhibin A with serum or plasma obtained from early or late pregnancy showed no evidence of cleavage, suggesting that 30K inhibin A is not the cleavage precursor. It is speculated that the formation of small molecular weight forms of both inhibin A and pro-αC is attributed to proteolytic changes, in part induced in the circulation during late gestation and in part by the placenta before secretion. It is concluded that inhibin A and pro-αC are processed in late pregnancy by more than one mechanism to form low molecular weight circulating forms of unknown structure.