The modulation of glucose uptake by cytosolic calcium and the role of insulin on calcium homeostasis in insulin-target cells are incompletely understood and results are contradictory. To address this issue, we used the C2C12 murine skeletal muscle cell line model and examined the influence of caffeine and 4-chloro-m-cresol, two ryanodine receptor agonists known to mobilize intracellular calcium stores and increase cytosolic free calcium concentration. We followed 45calcium efflux, a validated indicator of cytosolic calcium concentration, and 3-O-methyl-[1-3H]-d-glucose uptake in parallel. We also determined if insulin incubation affected 45calcium influx rate. A 30-min treatment by 1 microm insulin highly significantly increased 45calcium efflux by 8.5% (P = 0.0014), despite a significant reduction of 45Ca2+ influx already measurable after 20 and 30 min of insulin stimulation (-16.6%, P = 0.0119 and -21.3%, P = 0.0047, respectively). Caffeine (1-20 mm) and 4-chloro-m-cresol (0.05-10 mm) concentration-dependently increased 45calcium efflux, the latter being more potent and efficacious. These agents, in a concentration-dependent manner, inhibited both basal and, more potently, insulin-stimulated glucose uptake. This resulted in a negative correlation of glucose uptake and 45calcium efflux (r > 0.95, P < 0.001). This effect was approximately 5 times greater for caffeine than for 4-chloro-m-cresol, suggesting a calcium-independent part of the glucose uptake inhibition by caffeine. In our in vitro model of cultured muscle cells, insulin appears to prevent calcium overload by both stimulating efflux and inhibiting cell storage. This effect, taken together with the observed inhibitory, inverse relationship between 45calcium efflux and glucose uptake, contributes to describing the complex insulin-calcium interplay involved in target cells.