If newly formed adenosine is the mediator of active hyperemia in skeletal muscle, tissue adenosine must increase and remain elevated during sustained muscle contraction. We tested this prediction using isolated canine anterior calf muscles. Muscle samples were obtained before and during contraction by punch biopsy and adenosine was measured by spectrophotometric enzyme assay. Tissue adenosine content didnot increase significantly above the precontraction level during either the 2- or 6-Hz contraction. We estimated (by infusing adenosine) that the adenosine concentration required to cause a dilation equal to that observed during 6-Hz contractions was 3.7 X 10(-5) M. We would have detected an increase in tissue adenosine if this concentration were established in as little as 10% of the skeletal muscle interstitium during free flow. These results indicate that adenosine released into the whole interstitial space is not the cause of free-flow exercise hyperemia.