The vasodilating action of nitroglycerin (NTG) is thought to be mediated by its metabolic activation to nitric oxide (NO) in the vascular smooth muscle cell, but the site at which this process occurs has not been defined. To determine which cellular component is primarily responsible for this metabolic activation, subcellular fractions of bovine vascular smooth muscle cells were prepared and incubated with NTG along with cofactors. Time-dependent headspace NO concentrations generated in these preparations were determined directly by chemiluminescence detection. A mathematical model was developed to relate headspace NO with the NO-generating activity in each incubation, correcting for the concurrent processes of NO partitioning between the headspace and the incubation medium, and NO degradation in these two phases. The estimated NO-generating activities from different subcellular fractions were well correlated with the activities of two enzyme markers of the plasma membrane (K(+)-activated ouabain sensitive p-nitrophenyl phosphatase and 5'-nucleotidase), but not with those of the mitochondria, endoplasmic reticulum or the cytosol. These results indicate that the enzyme(s) responsible for the metabolic activation of NTG, and possibly other organic nitrate vasodilators, are associated with the plasma membrane in bovine coronary smooth muscle cells.