We surveyed pregnant women for macroprolactinemia and examined the heterogeneity of its etiologies. Serum samples obtained from 105 pregnant women (29.7 +/- 4.4 yr) during the third trimester were treated with polyethylene glycol, and 3 women (2.9%) were found to have a significantly high proportion of precipitated prolactin (PRL). Gel filtration studies revealed that big-big PRL (molecular weight greater than 100,000) was predominant (63.6, 74.0, 43.5% vs. 0.3 +/- 0.2% in normal pregnant women). The common clinical features of the 3 women included idiopathic hyperprolactinemia without any clinical symptoms such as amenorrhea and galactorrhea before pregnancy and very high levels of PRL (1680, 793, and 790 micrograms/L vs. 315 +/- 112 micrograms/L in normal pregnant women) during pregnancy. However, the nature of big-big PRL was different. Two of the 3 women possessed anti-PRL autoantibody (125I-PRL binding to the serum: 17.6%, 18.6% vs. 6.9 +/- 1.6% in normal pregnant women), but the other one did not have it. A significantly high proportion of PRL was absorbed to a concanavalin A column (41.1% vs. 4.0 +/- 2.1% in normal pregnant women), repetitive freezing and thawing of isolated big-big PRL resulted in a partial conversion to big and little PRL, and reduction of the isolated big-big PRL with 2-mercaptoethanol almost completely converted big-big PRL to little PRL in this woman. These findings suggest that this woman had a heterogeneous complex of covalently and noncovalently bound form of PRL with increased glycosylation. We conclude that macroprolactinemia is present with considerable frequency in pregnant women and that different etiologies are involved to form big-big PRL in macroprolactinemia.