Sixty-one sera from different trimesters of pregnancy were analyzed by two 125-I-NIH-HCG assay systems, employing anti-intact HCG serum and anti θ-HCG serum, respectively. The ratios of the levels measured in the two assay systems changed with the duration of pregnancy. The ratios during trimesters 1,2, and 3 were 2.94, 1.99, and 2.37, respectively. The cross-reactivity of proteohormones other than HCG was tested in both the assay systems. The two assay systems could be comparable in their high degree of specificity. However, the relative affinities of intact HCG and θ-HCG in the two assay systems were observed to be different. It was suggested that the significant differences in the ratios of the levels measured by the tow assay systems might have been infulenced by the occurrence of θ-HCG in serum and that the levels of the subunit must have changed at different stages of pregnancy