Stromal cells prepared from adipose tissue of women were maintained in monolayer culture to study the regulation of aromatase activity by hormones. Aromatase activity was stimulated 20-100-fold by dexamethasone. Half-maximal stimulation of aromatase activity was attained at a dexamethasone concentration of 2.5 nM. The stimulatory effect of dexamethasone was apparent after a preincubation time of 4 h, and stimulation was maximal after 24 h of preincubation. The stimulatory effect of dexamethasone was observed only when fetal calf serum also was present in the culture medium. Of the various steroids tested, dexamethasone was the most potent in stimulating aromatase activity. Cytosolic fractions prepared from stromal cells that had been maintained in monolayer culture were found to contain a homogeneous population of sites that specifically bound [3H]-dexamethasone with relatively high affinity (Kd = 2.9 nM) and low capacity (38 fmol/mg of protein). The stimulatory effect of dexamethasone on aromatase activity was prevented by simultaneous incubation with cortisol 21-mesylate (0.1-10 microM), a compound known to block the binding of glucocorticosteroids to cytoplasmic receptors. These findings are suggestive that glucocorticosteroids act to increase aromatase activity in stromal cells by binding to a glucocorticosteroid receptor. This presumably results in the induction of synthesis of specific proteins, perhaps the form of cytochrome P-450 involved in aromatization, or else NADPH-cytochrome P-450 reductase. Aromatase activity was also stimulated by dibutyryl cyclic AMP ( (Bu)2cAMP). In contrast to the stimulation by dexamethasone, the effect of (Bu)2cAMP was diminished by 70% when fetal calf serum (15%) was present in the culture medium. Aromatase activity of cells incubated with (Bu)2cAMP continued to increase over an 8 day period, at which time, enzyme activity was increased by 200-fold. Aromatase activity of stromal cells maintained in the presence or absence of serum was unaffected after incubation for 5 days with oFSH, hCG, bPRL, hGH or soterenol. On the other hand, incubation of cells for 5 days with cholera toxin (10 micrograms/ml) or 1-methyl-3-isobutylxanthine (0.1 mM) resulted in a marked increase in aromatase activity; the effect of these agents was diminished greatly when serum was present in the culture medium. The findings that serum inhibited the stimulation of aromatase activity by (Bu)2cAMP and that the time courses of aromatase induction by (Bu)2cAMP and dexamethasone were markedly different are suggestive that different molecular events mediate aromatase induction by glucocorticosteroids and cyclic AMP.