Embryos (n = 38) from an in vitro fertilization (IVF) program, unsuitable for transfer or cryopreservation, were maintained in culture for 14 days. The time course of hCG secretion into culture fluid was compared to observed embryo development. The earliest significant hCG rise was detected at day 7 post insemination for a partially hatched blastocyst, with hCG levels increasing 50-fold following blastocyst attachment to the surface of the culture vessel. Secretion of hCG was amplified by the addition of serum. There was no significant free alpha hCG or beta hCG secretion between days 6-12. We conclude that hCG production depends on the pathway of cytotrophoblast differentiation and commences with the appearance of the G1 phase in embryonic cells at the late blastocyst stage.