We used -escin-permeabilized pig cerebral microvessels (PCMV) to study the organization of carbohydrate metabolism in the cytoplasm of vascular smooth muscle (VSM) cells. We have previously demonstrated (Lloyd PG and Hardin CD. Am J Physiol Cell Physiol 277: C1250-C1262, 1999) that intact PCMV metabolize the glycolytic intermediate [1-¹³C]fructose 1,6-bisphosphate (FBP) to [1-¹³C]glucose with negligible production of [3-¹³C]lactate, while simultaneously metabolizing [2-¹³C]glucose to [2-¹³C]lactate. Thus gluconeogenic and glycolytic intermediates do not mix freely in intact VSM cells (compartmentation). Permeabilized PCMV retained the ability to metabolize [2-¹³C]glucose to [2-¹³C]lactate and to metabolize [1-¹³C]FBP to [1-¹³C]glucose. The continued existence of glycolytic and gluconeogenic activity in permeabilized cells suggests that the intermediates of these pathways are channeled (directly transferred) between enzymes. Both glycolytic and gluconeogenic flux in permeabilized PCMV were sensitive to the presence of exogenous ATP and NAD. It was most interesting that a major product of [1-¹³C]FBP metabolism in permeabilized PCMV was [3-¹³C]lactate, in direct contrast to our previous findings in intact PCMV. Thus disruption of the plasma membrane altered the distribution of substrates between the glycolytic and gluconeogenic pathways. These data suggest that organization of the plasma membrane into distinct microdomains plays an important role in sorting intermediates between the glycolytic and gluconeogenic pathways in intact cells.