Phosphorus-31 nuclear magnetic resonance (NMR) spectra (103.2 MHz) were obtained from rabbit portal vein, urinary bladder, and taenia coli smooth muscle. The muscles were mounted isometrically as strips in a horizontal probe and superfused with oxygenated medium at 23 degrees C. Under these conditions the preparations could remain in a stable metabolic state for at least 24 h. The resonances observed represented phosphocreatine (PC), adenosine triphosphate (ATP), and phosphomonoester. The PC-ATP ratio in relaxed preparations was approximately 1.9 (bladder) and 1.4 (taenia coli), approximately consistent with chemical analysis of tissue extracts by isotachophoresis. However, the levels of adenosine diphosphate (ADP) and inorganic phosphate (Pi) were considerably lower as estimated by NMR, suggesting intracellular compartmentalization. Contraction for 30 min in high-K+ medium caused a reversible 10-30% decline of PC but no change in ATP. Intracellular pH was 7.0 +/- 0.1 and was unchanged by contraction or inhibition of tissue respiration by cyanide. Treatment with 2-deoxyglucose resulted in accumulation of 2-deoxyglucose 6-phosphate, verifying the pH assignment. However, the phosphomonoester resonance of normal spectra is not glucose 6-phosphate. Treatment with Mg2+-free, high-Mg2+ (10 mM), or hyperosmotic media did not alter the Mg2+ saturation of ATP. The results obtained by the nontissue-destructive 31P-NMR measurements are consistent with the view that metabolic steady-state conditions are maintained during contraction in the smooth muscle.