We have found that in rat thymocytes binding of [¹²⁵I]choleragen is followed by cellular accumulation of cyclic 3′,5′-AMP which, in turn, is followed by stimulation of amino acid transport. Binding of cholera toxin was complete by 30 min and remained constant for the subsequent 150 min. After stimulation by choleragen, cellular cyclic 3′,5′-AMP became maximal by 30 min, after which it declined steadily so that by 90 min of incubation, cellular cyclic nucleotide levels were only 20% of those seen at 30 min. Stimulation of amino acid transport, although detectable by 15 min, did not become maximal until 120 min (by which time cellular cyclic 3′,5′-AMP had decreased by more than 80%). We have also used this system to delineate the step at which various pharmacologic agents and hormones act to alter the sequence of events mediating the response of rat thymocytes to cholera toxin. The ability of cycloheximide to abolish choleragen-stimulated amino acid influx without reducing [¹²⁵I]choleragen binding or cellular cyclic 3′,5′-AMP suggests that cyclic nucleotide stimulation of amino acid transport includes a step involving protein synthesis.