Sustained elevations in cytosolic calcium concentrations ([Ca2+]i) have been shown to render insulin target cells resistant to insulin action. In this study we examined the mechanisms of the detrimental effect of high levels of [Ca2+]i on insulin-induced 2-deoxyglucose (2-DOG) uptake. To elevate [Ca2+]i, we incubated rat adipocytes with either 40 mM potassium (K+) or 20 ng/ml PTH for 1 h for in vitro experiments and injected rats with PTH (injections of 50 micrograms, ip, every hour for 3 h) for in vivo studies. Adipocytes with elevated [Ca2+]i demonstrated a 30% decrease in insulin-stimulated 2-DOG uptake. A calcium channel blocker (nitrendipine) and a cAMP antagonist (RpcAMP) each partially restored insulin-stimulated glucose transport, but together they completely restored 2-DOG uptake. Concomitantly, we found a significant increase in phosphorylation of GLUT-4 in adipocytes with elevated [Ca2+]i. This change in GLUT-4 phosphorylation was also attenuated by nitrendipine and RpcAMP. These observations confirm that elevated [Ca2+]i diminishes insulin-stimulated glucose transport and suggest that increased phosphorylation of GLUT-4 in adipocytes with high [Ca2+]i may alter its intrinsic activity.