The purpose of the present investigation was to study the mode of action of a crude aqueous pineal extract (CAPE) on corticosterone (B) production from ACTH-mediated isolated adrenal cortex cells. Corticosterone production from a heterogenous adrenal cortex cell population, isolated from 8 male Sprague-Dawley rats, was measured fluorimetrically. CAPE (25 µl) was tested in this system using ACTH (0–5,000 pg/ml) and dibutyryl-c-AMP (0–100 nA//ml) as stimuli for a period of 1 h. In a separate experiment, CAPE (25 µl) was administered to ACTH (50 pg/ml) stimulated adrenal cortex cells for 15, 30, 60, and 120 min incubation periods. CAPE significantly decreased B produced by adrenal cortex cells at all doses of ACTH administered. CAPE also decreased the B produced by adrenal cortex cells when dibutyryl-c-AMP was used as a stimulus. The inhibitory effect of CAPE was manifest at some point in time between 30 and 60 min. It was significant at 60 min and highly significant at 120 min. It is evident from these data that CAPE and ACTH are not competing for the same receptor site.