The cultured human choriocarcinoma cell line, JEG-clone 3, secretes both biologically active hCG (JEG-hCG) and substantial quantities of free a-subunit (JEG-α). This study is concerned with a comparative characterization of the a-subunits of three distinct origins: 1) standard hCG-α obtained after dissociation of urinary (pregnancy) hCG, 2) JEG-α freely secreted by the JEG-3 cells, and 3) JEG-hCG-α obtained after dissociation of secreted JEG-hCG. Comparison of these a-subunits was made by gel exclusion chromatography, isoelectric focusing, and ability to form a radioreceptor assay-active hCG when incubated with standard hCG-β subunit. The freely secreted JEG-α exhibits an apparent molecular weight larger than that of standard hCG-a and JEG-hCG-α, whose apparent molecular weights are similar. The majority of JEG-α is represented by a component with acidic isoelectric pH (pI 4.8), and this component is virtually absent in standard hCG-α and JEG-hCG-α. When incubated with β-subunit, JEG-hCG-α forms a radioreceptor assay-active hCG almost to the same extent as does standard hCG-α, unlike JEG-α which is unable to do so. Thus, no major differences appear between the two α-subunits that were associated with a β-subunit [either in urinary (pregnancy) hCG or in JEG-hCG]. In contrast, different characteristics were found in freely secreted JEG-α. Our results indicate that two different types of α-subunit can be obtained from the JEG-3 cells: an α-subunit which is “normal”and incorporated into JEG hCG and another α-subunit which is freely secreted. Further understanding of the structural and chronological relation between these two different α-subunits obtained from the JEG-3 cells should prove valuable in the unraveling of hCG biosynthesis. © 1979 by The Endocrine Society.