Unsaturated long chain fatty acids modulate hormone secretion from a variety of endocrine glands, including the adrenal cortex. Oleic acid and linoleic acid have been shown to stimulate production of glucocorticoids in the absence of adrenocorticotropic hormone, but at a high concentration appeared to inhibit the action of this hormone. In the present study, the concentration dependence of the inhibitory actions of these two fatty acids was tested in collagenase-dispersed rat adrenal fasciculata cells, and the effects of both lipids on cAMP production were also determined. Adrenocorticotropic hormone stimulated steroid production from isolated cells approximately ten-fold above unstimulated cells. Oleic and linoleic acid significantly inhibited the response to this hormone by 44% and 54%, respectively. The half-maximally effective inhibitory concentration of both lipids was between 75-100 microM. A maximal concentration of adrenocorticotropic hormone increased cAMP secretion 138-fold above unstimulated cells. Oleic and linoleic acids inhibited the increase in cAMP secretion by 47% and 33%, respectively. It is concluded that pathophysiological concentrations of unsaturated fatty acids inhibit the action of adrenocorticotropic hormone on the adrenal gland, and that the mechanism of action of the lipids may be partly via inhibition of cAMP production.