Previous work by our laboratory has shown that glycolysis and gluconeogenesis occur in separate compartments of the vascular smooth muscle (VSM) cell and that an intact plasma membrane is essential for this compartmentation to exist. Disruption of the caveolae membrane domain by cyclodextrin inhibited glycolysis but stimulated gluconeogenesis, indicating that a link between caveolae and glycolysis may exist. Thus, we hypothesized that glycolytic enzymes are specifically localized to caveolae. To test this hypothesis we used confocal microscopy to determine the pattern of localization of caveolin-1 (CAV-1; caveolae-specific protein) and phosphofructokinase (PFK; glycolytic-specific protein) in freshly isolated VSM cells and cultured A7r5 cells. In freshly isolated cells we found that CAV-1 has a peripheral (membrane) localization and that most of the observed CAV-1 (85.3% ± 2.8) overlapped with PFK. However, only 59.9% ± 4.4 of PFK was localized with CAV-1 indicating a wider distribution of PFK than CAV-1. Cultured A7r5 cells exhibited compartmentation of glycolysis and gluconeogenesis and displayed two apparent phenotypes (contractile and proliferative) of smooth muscle cells distinguishable by shape. In both phenotypes of cultured A7r5 cells, CAV-1 fluorescence overlapped with PFK fluorescence (contractile phenotype= 83.1% ± 4.0, proliferative-phenotype= 81.5% ± 2.7). However, the overlap of PFK with CAV-1 was lower in the proliferative phenotype (35.9% ± 2.1) than the contractile phenotype (53.7% ± 4.3). Colocalization at particular sub-cellular regions indicated a progressive shift in pattern of colocalization from a primarily peripheral (membrane) localization in contractile cells to a primarily cytoplasmic localization in proliferative cells. Overall, cellular colocalization of PFK with CAV-1 was significant in all cell types (0.68 R² 0.77). Finally, since colocalization of PFK and CAV-1 occurred at the plasma membrane as well as at different cytoplasmic regions it suggests that PFK and CAV-1 might interact physically, regardless of the localization. Immunoprecipitation studies demonstrated that PFK co-immunoprecipitates with CAV-1 validating the possible interaction between the proteins. We conclude that a similar distribution of one pool of PFK with CAV-1 contributes to the compartmentation of glycolysis from other metabolic pathways and may provide for localized energetic support of kinases within the caveolae.