Nonimmune complexes of pregnancy-specific beta 1-glycoprotein (SP1) explain its heterogeneity. Two proteins reacting with antisera specific to pregnancy-specific beta 1-glycoprotein (SP1) have previously been detected in serum from pregnant women by use of crossed immunoelectrophoresis (CIE). The major SP1-reactive protein, SP1 beta, has a molecular weight of about 90,000 and the minor, antigenically deficient protein, SP1 alpha, about 200,000. In this study, gel filtration of serum on Ultrogel AcA 34 and Sephacryl S-300 has revealed that classical radioimmunoassay (RIA) techniques of two types only measure SP1 beta. With RIAs specially designed to detect complexed SP1 two higher molecular weight, SP1-containing complexes could be identified. CIE of serum or fractions from gel filtrations revealed a loss of the anodal part of the SP1 precipitate. However, when 4% polyethylene glycol (PEG) was incorporated in the anti-SP1-containing gel an antigenically deficient precipitate developed in the alpha-region. Incorporation of antialbumin in an intermediate gel in CIE reduced the anodal loss of the SP1 precipitate in anti-SP1-containing gels without PEG which resulted in a homogeneous peak. In addition, in anti-SP1-containing gels with PEG the anodal precipitate was reduced. This SP1-albumin complex fulfills all criteria for the previously described SP1 alpha. For measuring low levels of SP1 RIAs can be used. For higher values nephelometry is preferable. Although the amount of complexed SP1 is small in comparison with the amount of native SP1 methods employing electrophoresis are not recommended. In such tests SP1 complexed to fast-moving proteins like albumin might influence the results.