Concentrations of free cytoplasmic Ca2+ in rat aortic smooth muscle (RASM) cells were monitored using the ratiometric Ca2+ indicator fura 2- acetoxymethyl ester (AM). In RASM cells cultured in 5 mM Glc, incubation with angiotensin II, ATP, or thapsigargin [a selective inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase] depleted SR Ca2+ stores and initiated a capacitative Ca2+ influx through the plasma membrane. This influx was resistant to verapamil, a selective inhibitor of L-type voltage- gated Ca2+ channels, but was sensitive to SKF-96365, an inhibitor of the receptor-operated Ca2+ entry pathway. RASM cells cultured in 25 mM Glc exhibited a significant decrease in cytoplasmic Ca2+ responses to agonist- induced Ca2+ release from SR stores and to subsequent capacitative Ca2+ entry. In addition, the cytoplasmic response to thapsigargin-induced release of Ca2+ from the SR in hyperglycemic cells peaked more sharply than in control cells and returned to baseline more rapidly. The effects of hyperglycemia were not overcome by myo-inositol supplementation.