In the present experiment, we studied the inhibitory mechanism of Na+ depletion on high K(+)-induced contraction by simultaneously measuring reduced pyridine nucleotides (PNred) or oxidized flavoproteins (FPox) fluorescence and contractile tension of the guinea pig ileal longitudinal muscle. Tension, PNred and FPox were all reversibly increased by the addition of hyperosmotic 65 mM KCl (H-65K+). A high K+, Na(+)-deficient (Iso-154K+) solution induced a contraction followed by a gradual relaxation and gradually decreased PNred fluorescence. A hyperosmotic addition of NaCl to the Iso-154K+ solution prevented the decreases in tension and PNred fluorescence. Addition of pyruvate or oxaloacetate restored the decrease in Iso-154K(+)-induced contraction, but not the decrease in PNred fluorescence. In contrast to the PNred fluorescence, an application of the Iso-154K+ solution increased the FPox fluorescence which was not significantly changed by an addition of NaCl, pyruvate or oxaloacetate. These results suggest that the inhibitory mechanism of Na+ depletion on the Iso-154K(+)-induced contraction is an inhibition of glucose utilization.