A bioluminescent method is presented that allows monitoring of ATP production from mitochondria corresponding to one islet of Langerhans per sample. In mitochondria from ob/ob mice Ca2+ stimulates the ATP production in the presence of L-glycerol 3-phosphate (GP) by reducing the Km for GP by one order of magnitude to about 3 mM. Maximal ATP production in the presence of Ca2+ (200 nM) is obtained at 10 mM GP. The free calcium concentration required to reach half-maximal stimulation (K0.5Ca2+) depends on the GP concentration, thus half-maximal effects are observed at about 80 nM at low GP (1 mM) and 10 nM at high GP (10 mM). Sodium can replace Ca2+ as a stimulator of GP-induced ATP production. It activates ADP phosphorylation by B-cell mitochondria in a sigmoidal concentration-dependent manner in the absence of Cs2+ (Hill coefficient 2.3 +/- 0.2) but does not change K0.5ca2+ nor the maximal mitochondrial activity. Ca2+ concentrations higher than 300 nM are inhibitory at all tested substrate concentrations. Mitochondria from ob/ob mice showed no functional defect when compared with normal controls. It is concluded that activation of the glycerol phosphate shuttle may not be the main coupling site for glucose-induced insulin release at maximal cytoplasmic Ca2+ levels.