Cytosol prepared from rat epididymal fat cells by centrifugation at 100,000 X g for 1 hr was found to enhance the basal and epinephrine-sensitive adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] of fat cell ghosts. Cholera toxin also stimulated adenylate cyclase and increased the response to epinephrine in fat cells. A possible relationship between the adenylate cyclase modifying activities of cytosol and the effects of cholera toxin was sought. Cytosol from freshly prepared fat cells added to ghosts prepared from cells that had been exposed to toxin for varying periods showed a progressive loss of responsiveness to cytosol epinephrine-enhancing activity. The effect appeared within 15 min after toxin exposure, a full 30 min before any direct effect of toxin on adenylate cyclase was seen. Since exposure to toxin decreased membrane response to cytosol epinephrine-enhancing activity, the possibility that epinephrine-enhancing activity in cytosol might be altered by toxin was explored. Cytosol from cells exposed to toxin for varying periods lost epinephrine-enhancing activity to an appreciable degree within 15 min. Examination of these early events after exposure to toxin should clarify the way in which this bacterial substance affects mammalian cells. The cytosol epinephrine-enhancing activity was destroyed by boiling for 3 min and was partially inactivated by trypsin. It was nondialyzable and stable at -70 degrees.