We used ¹³C-labeled substrates and nuclear magnetic resonance spectroscopy to examine carbohydrate metabolism in vascular smooth muscle of freshly isolated pig cerebral microvessels (PCMV). PCMV utilized [2-¹³C]glucose mainly for glycolysis, producing [2-¹³C]lactate. Simultaneously, PCMV utilized the glycolytic intermediate [1-¹³C]fructose 1,6-bisphosphate (FBP) mainly for gluconeogenesis, producing [1-¹³C]glucose with only minor [3-¹³C]lactate production. The dissimilarity in metabolism of [2-¹³C]FBP derived from [2-¹³C]glucose breakdown and metabolism of exogenous [1-¹³C]FBP demonstrates that carbohydrate metabolism is compartmented in PCMV. Because glycolytic enzymes interact with microtubules, we disrupted microtubules with vinblastine. Vinblastine treatment significantly decreased [2-¹³C]lactate peak intensity (87.8 ± 3.7% of control). The microtubule-stabilizing agent taxol also reduced [2-¹³C]lactate peak intensity (90.0 ± 2.4% of control). Treatment with both agents further decreased [2-¹³C]lactate production (73.3 ± 4.0% of control). Neither vinblastine, taxol, or the combined drugs affected [1-¹³C]glucose peak intensity (gluconeogenesis) or disrupted the compartmentation of carbohydrate metabolism. The similar effects of taxol and vinblastine, drugs that have opposite effects on microtubule assembly, suggest that they produce their effects on glycolytic rate by competing with glycolytic enzymes for binding, not by affecting the overall assembly state of the microtubule network. Glycolysis, but not gluconeogenesis, may be regulated in part by glycolytic enzyme-microtubule interactions.