The immunoreactive insulin (IRI) release patterns produced by continuous theophylline stimulation of rat pancreas have been defined, using an in vitro perfusion system. In the presence of glucose, citrate, and pyruvate at concentrations which were nonstimulatory by themselves, continuous stimulation with theophylline produced a biphasic IRI release profile. In the absence of substrate, continuous theophylline stimulation produced only an abrupt and limited primary response. Of the substrates tested, only glucose significantly enhanced this primary response. With increasing theophylline concentrations, whether in the presence or absence of substrate, significant increases were noted in the primary response as estimated by either the maximum rate of IRI release attained or by the total amount of IRI released during this time. Similarly, the secondary responses to theophylline increased with theophylline concentration in the presence of either citrate or pyruvate. With glucose as substrate, however, increasing theophylline concentrations from 2.5 to 5, then 10 mM produced a progressive reduction in both indices of the secondary response, which was inversely related to the primary response. These findings suggest that cyclic AMP not only mediates IRI release in quantitative terms but is also implicated in the qualitative nature of the response pattern. They also indicate a possible metabolic basis for biphasic IRI release, the acute or primary response being dependent upon the basal state of the cell and the availability of endogenous energy sources, the secondary response upon the availability of exogenous substrate.