We have analysed the structure and composition of the β-core fragment of human chorionic gonadotrophin (βC-hCG) from fresh urine specimens obtained from pregnant women and compared our findings with those previously proposed by other groups using different protocols. SDS-PAGE separation of reduced βC-hCG demonstrated two major bands with apparent molecular weights of Mr 8900 and Mr 7500. The molecular weight of the agalacto βC-hCG was estimated to be Mr 10 218 from the amino acid analysis after high-performance liquid chromatography (HPLC) separation. Moreover, HPLC separation of its reduced and S-carboxymethylated peptides resulted in three peaks, but only two of them could be sequenced and demonstrated to be the previously reported β6-40 (Mr 5000) and β55-92 (Mr 5300) peptides of the βhCG subunit. The results showed that 56-78% of βC-hCG molecules of molecular weight Mr 12 800 were able to bind Concanavalin A (Con A). While most were lacking all the peripheral monosaccharides and terminated in mannose, some retained other sugar residues on their antennae. Direct carbohydrate analysis showed the following molar content normalized to six mannose molecules: galactose 2·8, glucosamine 5·3, galactosamine 0·3, fucose 1·7 and sialic acid 3·0. Approximately 22–44% of the βC-hCG molecules did not bind Con A (Con A non-reactive forms), of which 88% were totally deprived of sugar units and had an apparent molecular weight of approximately Mr 10 000, and 12% were weakly reactive to Con A and reactive to anion exchange (negatively charged forms), being incompletely trimmed of their oligosaccharide chains. Comparison of our results with those of two other groups have indicated that the differences noted among preparations are due to either the source or the methods used to purify and characterize this fragment. In addition, our results showed significant microheterogeneity on the N-linked oligosaccharide moieties with some molecules apparently having no sugar molecules. These results have implications for the origins of βC-hCG, suggesting secretion of some molecules without sugar chains and in other cases possible metabolism of hCG in the peripheral tissues. Journal of Endocrinology (1993) 139, 519–532