The contribution of adenosine to blood flow autoregulation in striated muscle was evaluated by direct in vivo visualization of arterioles in the rat cremaster muscle. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium, and the cremaster muscle was surgically exposed and maintained in a controlled tissue bath environment with pH 7.40, CO2 tension (PCO2) congruent to 40 mmHg, and O2 tension (PO2) at either a high (congruent to 70 mmHg) or a low (congruent to 10 mmHg) value. Local adenosine activity was blocked in some animals by the addition of theophylline (3 X 10(-5) M) to the bath medium. Individual second (2A)- and third (3A)-order arterioles were observed via closed-circuit television microscopy, and blood flow in each arteriole was calculated from simultaneous measurements of arteriolar diameter and red blood cell velocity. Perfusion pressure to the animal's hindquarters was altered by varying the degree of occlusion of the sacral aorta; arteriolar diameter, velocity, and blood flow responses were plotted as a function of the varying pressure. Both 2A and 3A arterioles exhibited vasodilation and substantial superregulation of blood flow (increased blood flow with decreased perfusion pressure) when bath PO2 was low and adenosine activity was not blocked. Addition of theophylline to the cremaster bath medium significantly reduced the dilation and abolished superregulation, although substantial autoregulation remained. When bath PO2 was high, the degree of arteriolar dilation and autoregulation was reduced compared with the low bath PO2 responses, and blocking adenosine activity had no effect on the responses. These results support the concept that changes in local adenosine levels are involved in the autoregulatory responses observed in the rat cremaster muscle and that the magnitude of adenosine's contribution is directly related to the degree of tissue hypoxia. However, blocking adenosine activity did not totally abolish autoregulation, suggesting that other metabolic and/or myogenic factors may also be contributing to blood flow regulation in this tissue.