H2 clearance is a recently described method of measuring gastric mucosal blood flow that has great potential for clinical use. However, the effects of luminal pH and of secretory activity of the gastric mucosa on the accuracy of H2 clearance measurements have not been systematically examined. We therefore tested the validity of H2 clearance measurements at different pHs in both in vitro and in vivo systems. In addition, we compared measurements by H2 clearance and radioactive microspheres during stimulation and suppression of acid secretion. In vitro, H2 washout was relatively constant over a range of pHs from 2.0 to 8.0. In chambered segments of canine fundus in vivo, H2 clearance was not significantly affected by pH of the luminal solutions either in the resting state or at lower blood flows induced during infusion of vasopressin. Finally, there was a close correlation (r = 0.85; p less than 0.001) between H2 clearance and microsphere measurements under resting conditions, during intravenous histamine stimulation, and after infusion of cimetidine to suppress acid secretion. In summary, H2 clearance reliably and accurately measures gastric mucosal blood flow at different luminal pHs and under conditions that stimulate or suppress acid secretion.