The aim of this study was to determine whether the pathways of glycolysis and glycogenolysis can be independently modulated by the provision of acetate or pyruvate as exogenous substrates. Hog carotid artery segments were allowed to replete glycogen stores to over 6 micromol/g of new 13C-labeled glycogen by incubation at 37 degrees C with 5 mM [1-13C]glucose for 6-16 h and then were isometrically contracted for 3 h with 80 mM KCl in the presence of 5 mM [2-13C]glucose and either 2 mM sodium acetate or 5 mM sodium pyruvate. Measurements were made of total lactate production, glucose utilization, glycogen utilization, isometric force, [2-13C]lactate and [3-13C]lactate production. Compared to experiments with glucose as the sole exogenous substrate, provision of pyruvate significantly decreased glucose utilization (by 28%) but insignificantly decreased glycogen utilization. In contrast, provision of acetate resulted in a statistically insignificant decrease in glucose utilization (by 23%) and an increase in glycogen utilization (by 20%). The fraction of [3-13C]pyruvate derived from glycogen that was converted to [3-13C]lactate was significantly decreased in the presence of acetate despite the enhanced glycogen utilization. Despite these alterations in cellular energy balance, isometric force generation and maintenance was similar for all experimental groups. This differential regulation of glycolysis and glycogenolysis may either reflect the compartmentation of these pathways or suggest a novel regulation of carbohydrate metabolism in vivo.