Objectives:To determine the accuracy of intramucosal pH (pHi) calculated using arterial bicarbonate instead of mucosal capillary bicarbonate in the Henderson-Hasselbalch equation.Design:Simulation of progressive ischemia in mucosal capillary blood.Setting:University research laboratory.Subjects:Normal human blood diluted with plasma.Interventions:Three venous blood specimens were heparinized and diluted to a mean hemoglobin concentration of 5.0 (±0.9) g/dL by addition of plasma (2:1, vol:vol). Mucosal capillary aerobic flow stagnation was simulated by multiple exposures of each cooled specimen to a gas mixture containing 90% nitrogen and 10% CO2. When PCO2measured at 37°C (98.6°F) was approximately 120 torr (16 kPa), the assigned anaerobic threshold, subsequent anaerobic flow stagnation was simulated by mixing the hypercapnic specimens in sealed syringes with five to six successive small aliquots (<100 μL) of lactic acid (10 g/L).Measurements and Main Results:The relationship between PCO2and pH in the specimens was compared with the relationship between the same PCO2values and pHi calculated by substituting bicarbonate concentrations of 22 and 26 mmol/L in the Henderson-Hasselbalch equation. As PCO2rose from 50 torr (8 kPa), conventionally calculated pHi increasingly underestimated simulated mucosal capillary pH, with bias >0.1 pH unit at the simulated anaerobic threshold of 120 torr (16 kPa). As PCO2rose further the values converged, becoming equivalent at PCO2∼ 150 torr (20 kPa). From PCO2≥ 200 torr (26.7 kPa), conventional pHi progressively overestimated simulated mucosal pH. The difference was >0.3 pH units at PCO2= 250 torr (33.3 kPa).Conclusions:In the mucosal PCO2range usually encountered clinically, the arterial bicarbonate substitution causes underestimation of mucosal capillary pH. With moderate mucosal capillary lactic acidosis the error becomes small, and in severe regional ischemia there is significant overestimation of mucosal capillary pH.