Stimulation of insulin release by glucose is widely thought to be coupled to a decrease in the activity of ATP-sensitive K+ channels (KATP channels) that is caused by a decreased concentration of free ADP. To date, most other investigators have reported only on total cellular ADP concentrations, even though only a small fraction of all ADP is free and only the free ADP affects KATP channels. We tested the hypothesis that amino acids elicit insulin release via a decrease in the activity of KATP channels owing to a decrease in the level of free ADP. We estimated the concentration of free ADP in betaHC9 hyperplastic insulin-secreting cells based on the cell diameter and on luminometric measurements of ATP, phosphocreatine, and total creatine. The concentration of free ADP fell exponentially as the concentration of glucose increased. A physiological mixture of amino acids greatly stimulated insulin release at 0-30 mmol/l glucose but affected the concentration of free ADP only to a minor degree and significantly so only at < or = 2 mmol/l glucose. In the presence of 2-deoxyglucose and NaN3, amino acids were unable to stimulate insulin release. When KATP channels were held open with diazoxide (and the plasma membrane partially depolarized with high extracellular KCl), amino acids still stimulated insulin release. We conclude that amino acid-induced insulin release depends on two components: a yet-unknown amino acid sensor and KATP channels, which serve to attenuate hormone release when cellular energy stores are low. We propose that glucose-induced insulin release may be regulated similarly by two components: glucokinase and KATP channels.