Current research in organ physiology often utilizes in situ or isolated perfused tissues. We have characterized a perfusion medium associated with excellent performance characteristics in perfused mammalian skeletal muscle. The perfusion medium consisting of Krebs–Henseleit buffer, bovine serum albumin, and fresh bovine erythrocytes was studied with respect to its gas-carrying relationships and its response to manipulation of acid–base state. Equilibration of the perfusion medium at base excess of −10, −5, 0, 5, and 10 mmol∙L−1 to humidified gas mixtures varying in their CO2 and O2 content was followed by measurements of perfusate hematocrit, hemoglobin concentration, pH, [Formula: see text], [Formula: see text], [Formula: see text], and percent oxygen saturation. The oxygen dissociation curve was similar to that of mammalian bloods, having a P50 of 32 Torr (1 Torr = 133.3 Pa), Hill's constant n of 2.87 ± 0.15, and a Bohr factor of −0.47, showing the typical Bohr shifts with respect to CO2 and pH. The oxygen capacity was calculated to be 190 mL∙L−1 blood. The carbon dioxide dissociation curve was also similar to that of mammalian blood. The in vitro nonbicarbonate buffer capacity (Δ[HCO3−] × ΔpH−1) at zero base excess was −24.6 and −29.9 mmol∙L−1∙pH−1 for the perfusate and buffer, respectively. The effects of reduced oxygen saturation on base excess and pH of the medium were quantified. The data were used to construct an acid–base alignment diagram for the medium, which may be used to quantify the flux of nonvolatile acid or base added to the venous effluent during tissue perfusions.