The synthesis and secretion of human chorionic gonadotropin (hCG) subunits have been studied by pulse-chase techniques in JAR choriocarcinoma cells, which produce both the alpha and beta subunits of hCG "eutopically," that is, as part of their expected repertoire of gene products, and in three cell lines that produce either alpha or beta subunit "ectopically." JAR cells contain two intracellular forms of the alpha subunit (Mr = 15,000 and 18,000) and of the beta subunit (Mr = 18,000 and 24,000) but do not accumulate fully processed, "mature" alpha (Mr = 22,000) or beta (Mr = 34,000) subunits during chase of pulse-labeled cells. Mature subunits, however, are secreted into the culture medium during this time. Thus, secretion of mature subunits appears to occur rapidly after processing of the intracellular forms. Two cell lines that ectopically secrete alpha but not beta subunit, ChaGo bronchogenic carcinoma cells and HeLa S3 cervical carcinoma cells, also contain the Mr = 15,000 and 18,000 intracellular forms of alpha subunit and appear to accumulate mature alpha subunit intracellularly. The CBT cell line, derived from a glioblastoma multiforme, produces the Mr = 18,000 and 24,000 intracellular forms of beta subunit with no evidence for alpha subunit synthesis or secretion. These four cell lines should provide "biologic reagents" for the further study of alpha and beta subunit processing and secretion.