To study the effects of -opioid receptor stimulation on intracellular Ca²⁺ concentration ([Ca²⁺]_i) homeostasis during extracellular acidosis, we determined the effects of -opioid receptor stimulation on [Ca²⁺]_i responses during extracellular acidosis in isolated single rat ventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dose dependently decreased the electrically induced [Ca²⁺]_i transient, which results from the influx of Ca²⁺ and the subsequent mobilization of Ca²⁺ from the sarcoplasmic reticulum (SR). U-50488H (30 µM) also increased the resting [Ca²⁺]_i and inhibited the [Ca²⁺]_i transient induced by caffeine, which mobilizes Ca²⁺ from the SR, indicating that the effects of the -opioid receptor agonist involved mobilization of Ca²⁺ from its intracellular pool into the cytoplasm. The Ca²⁺ responses to 30 µM U-50488H were abolished by 5 µM nor-binaltorphimine, a selective -opioid receptor antagonist, indicating that the event was mediated by the -opioid receptor. The effects of the agonist on [Ca²⁺]_i and the electrically induced [Ca²⁺]_i transient were significantly attenuated when the extracellular pH (pH_e) was lowered to 6.8, which itself reduced intracellular pH (pH_i) and increased [Ca²⁺]_i. The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potent Na⁺/H⁺ exchange blocker, or 0.2 mM Ni²⁺, a putative Na⁺/Ca²⁺ exchange blocker. The observations indicate that acidosis may antagonize the effects of -opioid receptor stimulation via Na⁺/H⁺ and Na⁺/Ca²⁺ exchanges. When glucose at 50 mM, known to activate the Na⁺/H⁺ exchange, was added, both the resting [Ca²⁺]_i and pH_i increased. Interestingly, the effects of U-50488H on [Ca²⁺]_i and the electrically induced [Ca²⁺]_i transient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca²⁺ (3 mM) solution was superfused, the resting [Ca²⁺]_i increased; the increase was abolished by 0.2 mM Ni²⁺, but the pH_i remained unchanged. Like the responses to superfusion with high-concentration glucose and extracellular acidosis, the responses of the [Ca²⁺]_i and electrically induced [Ca²⁺]_i transients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time that extracellular acidosis antagonizes the effects of -opioid receptor stimulation on the mobilization of Ca²⁺ from SR. Activation of both Na⁺/H⁺ and Na⁺/Ca²⁺ exchanges, leading to an elevation of [Ca²⁺]_i, may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.