A detailed understanding of adenosine metabolism of vascular smooth muscle cells (VSMC) is highly desirable to critically evaluate possible autocrine effects of adenosine in this cell species. Therefore, this study quantified intra- and extracellular adenosine flux rates, the transmembrane concentration gradient, and the adenosine surface concentration in porcine VSMC and, for comparison, aortic endothelial cells (PAEC). Cell-covered microcarrier beads packed in a chromatography column were superfused with a HEPES buffer. With the use of specific inhibitors of adenosine kinase (iodotubericidine, 10 µM), adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl)-adenine, 5 µM], ecto-5'-nucleotidase (,-methylene-adenosine 5'-diphosphate, 50 µM), and adenosine membrane transport (n-nitrobenzylthioinosine, 1 µM), total production rates of 12.3 ± 2.7 and 7.5 ± 1.3 pmol · min¹ · µl cell volume¹ were obtained for VSMC and PAEC, respectively. Despite prevailing intracellular adenosine production (76 and 70% of total production, respectively), transmembrane concentration gradients under control conditions were directed toward the cytosol as a result of rapid intracellular adenosine rephosphorylation and continuous extracellular hydrolysis from 5'-AMP. Surface concentrations were ~18 nM in VSMC and PAEC under control conditions and increased to ~60 nM during partial inhibition of adenosine metabolism. Simultaneously, the transmembrane adenosine concentration gradient was reversed. We conclude that adenosine flux rates in VSMC and PAEC are quantitatively similar and that VSMC may influence the interstitial adenosine concentration under basal steady-state conditions.