To determine if developmental variations existed in the second messenger system that mediates cholera toxin (CT) action, the adenylate cyclase (AC) response was studied in 2-wk-old suckling and 6-wk-old weaned rats. AC was assayed in the proximal small intestine 4 h after intraduodenal administration of various doses of CT. Dose-effect analysis showed a 9-fold increase in the sensitivity of the CT-activated cyclase response in suckling rats when measured by the ED50, expressed as microgram CT/g body wt (0.03 for 2 wk, 0.27 for 6 wk). When the CT dose was expressed as microgram/animal, suckling rats were 50 times more sensitive than 6-wk-old rats. In addition, the CT-induced fluid secretion was closely correlated with the elevated cyclase activities (correlation coefficient: 0.83 for 2 wk, 0.93 for 6 wk). Furthermore, more fluid seemed to be secreted/unit wt of gut in the sucklings, even when the same level of enzyme activity was compared. A maximum of 3- to 4-fold rise in AC activation occurred at 0.5 microgram CT/g body wt, but both the basal and the maximal stimulated levels of AC were not developmentally different. This study demonstrates an in vivo increase in AC responsiveness to CT that may be in part responsible for the increased incidence of toxigenic diarrhea in neonates.