The rat zona glomerulosa has a renin-angiotensin system that appears to function as an autocrine or paracrine system in the regulation of aldosterone production. To further investigate dynamic changes of production of renin and aldosterone in vitro we developed a primary monolayer culture of rat adrenal glomerulosa cells in serum-free medium. Collagenase- dispersed glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 48 hours; the medium was then replaced with serum-free PFMR-4 medium. The cell viability and the aldosterone secretion were stable over the additional 48 hours in the serum-free control medium. After incubation for 24 hours in the serum-free medium, the cells were exposed to high K+or adrenocorticotropic hormone (ACTH) for another 24 hours. ACTH stimulated aldosterone secretion, and this increased secretion was associated with an increase in renin activity (cell active renin, from 15.56±0.71 to 45.75±5.69; cell inactive renin, from 0.67±0.54 to 8.75±3.40; medium inactive renin, from 5.58±1.16 to 106.20±14.01 pg angiotensin I (Ang I)/μg protein/3 hr). Aldosterone was also stimulated by high K+. This increase was also associated with an increase in active renin in the cells (from 15.08±1.80 to 23.26±2.15 pg Ang I/μg protein/3 hr) and an increase in inactive renin in the medium (from 10.87±1.62 to 21.37±3.20 pg Ang I/μg protein/3 hr). Addition of the angiotensin converting enzyme inhibitor lisinopril attenuated both ACTH- and high K+-stimulated aldosterone secretion significantly. These data indicate that a local renin system may play a role in the regulation of aldosterone production.