The influence of acidosis on adenosine output from the isolated constant-flow-perfused gracilis muscle was studied in anesthetized dogs. Depression of intracellular pH (pHi) by supplementation of the inspired air with 10% CO2-90% O2 increased arterial PCO2 from 34.2 +/- 1.0 to 53.5 +/- 1.9 mmHg, arterial PO2 from 138.3 +/- 3.9 to 256.6 +/- 17.6 mmHg, and venoarterial adenosine concentration from 14 +/- 15 to 47 +/- 19 nM. Twitch contractions of the muscle at 2 Hz increased venoarterial adenosine concentration to 165 +/- 63 and 204 +/- 62 nM in normocapnia and hypercapnia, respectively. Venoarterial lactate concentration increased from 0.42 +/- 0.07 to 0.90 +/- 0.15 mM during normocapnic contractions but remained unchanged during hypercapnic contractions (0.42 +/- 0.11 mM). Depression of pHi by infusion of amiloride and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid increased venoarterial adenosine concentration from -2 +/- 27 to 124 +/- 48 nM in normocapnia and from 16 +/- 24 to 236 +/- 119 nM in hypercapnia. These results indicate that adenosine output from red oxidative skeletal muscle was stimulated by procedures that depress pHi.