The development of long term culture conditions with which to study the regulation of expression of aromatase, cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase (3βHSD) in human granulosa-lutein cells is described in this report. Conditions have been established for the dispersal, growth, freezing, and storage of functional human granulosa cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Optimal growth conditions for human granulosa-lutein cells were determined by plating cells at a low density and testing the capacity of a variety of culture conditions to support growth. A combination of fetal bovine serum (FBS), horse serum, and the serum substitute UltroSer G was found to increase cell number to maximal levels, 8- to 10-fold higher than with sera alone. Human granulosa-lutein cells grown under these conditions had a doubling rate of 36–40 h and were morphologically distinct from human theca interna cells grown under similar conditions. Human granulosa-lutein cells treated with forskolin retracted and rounded up, whereas cultures of human ovarian theca interna cells or human fibroblasts treated similarly did not retract. Human granulosa-lutein cells were grown for successive passages and transferred to serum-free medium containing forskolin, LH, hCG, or cholera toxin. Addition of these agents resulted in a time- and dose-dependent increase in aromatase activity and progesterone secretion. In these studies FSH treatment was found not to increase aromatase activity. In a study of the time course of 3βHSD activity in the absence of forskolin under serum-free conditions, it was found that 3βHSD activity increased 3-fold during the 72-h treatment period. Forskolin-stimulated 3βSHSD activity also increased in a time-dependent manner, with levels in treated cells 3-fold higher than those in control cells. Northern analysis performed on total RNA obtained from forskolin- or hCG-stimulated granulosa-lutein cells confirmed that the increase in aromatase activity was associated with a corresponding increase in levels of mRNA specific for aromatase cytochrome P-450. Levels of mRNA encoding cholesterol side-chain cleavage cyto-chrome P-450 were similarly increased in cells treated with forskolin compared with unstimulated values at each of the time points investigated. Under serum-free conditions in the absence of stimulation, the 3.4-kilobase band of aromatase cytochrome P-450 mRNA was detectable. After treatment with hCG or forskolin, two principal hybridizable mRNA species of 3.4 and 2.9 kilobases were observed in addition to a smaller species, as has been previously reported. No hybridizable 17α-hydroxylase cytochrome P-450 was detectable.The development of conditions under which human granulosa cells can be stored and subsequently propagated with maintenance of inducibility of characteristic steroidogenic enzymes, provides for a reproducible system for investigating the regulation of these enzymes by stimulatory and inhibitory factors.