The purpose of this study was to develop primary cultures of human chorion laeve cells and examine certain aspects of steroid metabolism during culture. Tissues obtained by elective cesarean section at term (38-40 weeks) were dispersed with collagenase. Cells were isolated on Percoll gradients at the interface between 20% and 40% Percoll and examined in primary culture for up to 1 week. Cultures were carried out in chemically defined media supplemented with 10% or 0.1% fetal calf serum (FCS). The morphological and biochemical properties of the cells were different in the two systems. In 0.1% FCS, cells formed clumps of tissue within 16 h of plating, and there was no cell replication. In contrast, in 10% FCS, the cells formed a carpet of tissue and reached confluence after 5 days in culture, resulting in increased DNA and protein content and thymidine incorporation in the dishes. Three steroidogenic enzymes were studied during culture: alkyl steroid sulfatase, estrogen sulfatase and 3 beta-hydroxysteroid dehydrogenase. The sulfatases had higher activities in 0.1% than in 10% FCS, and their activities decreased markedly during the culture period. In contrast, 3 beta-hydroxysteroid dehydrogenase activity was higher in 10% FCS than in 0.1% FCS. Activity remained constant during the culture period in 0.1% FCS and increased in 10% FCS. In the latter system this increase resulted in the enzyme maintaining a constant specific activity during culture. These studies describe two viable systems of chorion laeve cells in primary culture, which may be valuable for studying long term and/or subtle effects on various metabolic aspects of this tissue.