To elucidate the role of glycogen in the contraction of tracheal smooth muscle, we investigated the changes in the glycogen contents of the bovine trachea during contractions induced by high K(+) and hypoxia (achieved by bubbling N(2) instead of O(2)), either in a glucose-free condition or in the presence of iodoacetic acid (IAA), an inhibitor of glycolysis. Hyperosmotic addition of 65 mM KCl (H-65 K(+)) induced a sustained contraction. A glucose-free condition did not affect H-65 K(+)-induced contraction. However, hypoxia slightly inhibited the contraction, and glucose-free PSS with hypoxia or IAA remarkably inhibited the H-65 K(+)-induced contraction. H-65 K(+) induced a sustained increase in reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity. Hypoxia alone slightly enhanced PNred fluorescence, and when combined with a glucose-free condition, it remarkably enhanced the H-65 K(+)-induced PNred fluorescence. IAA inhibited PNred fluorescence. In the presence of H-65 K(+), a glucose-free condition, hypoxia and the combination of glucose-free PSS and hypoxia decreased the glycogen contents. However, IAA had no effect on glycogen contents. Although hypoxia or glucose-free PSS did not affect PCr and ATP contents, the combination of hypoxia and glucose-free PSS or IAA induced a gradual decrease of PCr content. In conclusion, we suggest that endogenous glycogen was utilized to increase the activity of glycolysis for maintaining high K(+)-induced contraction of the bovine trachea in the glucose -free and/or hypoxic condition.