Abstract We wanted to determine whether CO 2, H +, and K + affect the adenosine metabolism of vascular smooth muscle in a way that could account for the effects of these substances on vascular reactivity and their ability to modulate adenosine-induced vascular relaxation. Accordingly, 1-week-old cultures of rat aortic smooth muscle were incubated in phosphate-buffered saline with various [K +]'s and pH's and aerated in an incubation chamber with gases containing various proportions of CO 2. Uptake was measured as 14C incorporation into cellular constituents during exposure to 2μ M [ 14C]adenosine. Release was measured as net extracellular adenosine accumulation. Uptake of adenosine was not significantly affected by any of the experimental maneuvers, except that it was greatly attenuated by dipyridamole (10 −5 and 10 −4 M) and transiently enhanced by the low CO 2 levels. Adenosine release, however, was depressed by lowering atmospheric CO 2 (0% vs 5%) and also by normocapnic acidosis (pH 6.8 vs pH 7.4). We conclude that vascular smooth muscle in culture releases adenosine at a rate that might have vasoactive significance in vivo. Furthermore, some of the vascular actions of CO 2 and H +, but not those of K +, may be partially explained by their effects on vascular smooth muscle's adenosine metabolism.