Concomitant with glucose-induced insulin release, there occurred an increase of ATP from 4.40 plus or minus 0.21 to 23.16 plus or minus 0.52 pmol/100 islets/min (P less than 0.001) in the effluent from perifused rat islets. There is a linear relationship between circulating ATP and insulin levels both in the stimulated and basal state (r = 0.689, P less than 0.01). Islets incubated with labeled adenine for a short period of time (37.5 min) showed no release of radioactivity upon subsequent glucose-induced insulin release. Islets incubated for a prolonged interval with labeled adenine (150 min) showed an increase in acid soluble radioactivity in the effluent during glucose-induced insulin release. Following incubation of the islets with labeled adenine for 150 min, approximately 5% of the homogenate radioactivity was found in the secretory granules. Using column chromatography to separate the adenine nucleotides, the distribution of radioactivity among the various nucleotides in the secretory granule fraction was found to be: AMP 54.42 plus or minus 4.96%, ADP 14.20 plus or minus 1.63%, ATP 15.39 plus or minus 3.84%, and cAMP 16.07 plus or minus 2.11%. The distribution of radioactivity in the effluent adenine nucleotides after glucose-induced insulin release was: AMP 32.83 plus or minus 4.62%, ADP 24.52 plus or minus 2.77%, ATP 28.13 plus or minus 5.45%, and cAMP 26.01 plus or minus 3.34%. The absolute levels of adenine nucleotides in the secretory granules were ATP 4.19 plus or minus 0.88, ATP madp 7.94 plus or minus 2.20 and cAMP 4.46 plus or minus 1.74 pmol/ug prot. The levels in the islet effluent were ATP, 15.30 plus or minus 2.70, ATP qDP, 29.43 plus or minus 3.49 and cAMP 7.66 plus or minus 1.93 pmol/100 islets/min for the first ten min of glucose-stimulated insulin release. Thereafter there was a rapid decline in effluent cAMP while ATP and ADP remained in essentially equivalent amounts. The distribution of radioactivity and absolute levels of the adenine nucleotides in the effluent reflects that found in the secretory granules, confirming previous observations that insulin release is occurring by exocytosis.