In typical physiological solutions, CO2 is in equilibrium with HCO3- and H+ (CO2 + H 2O⇆HCO3- + H+). Because one cannot independently alter CO2 and HCO3- concentrations and pH, it is impossible to distinguish between the effects of CO2 and HCO3-on physiological processes. Here we describe a continuous-flow, rapid-mixing approach for generating out-of-equilibrium CO2/ HCO3- solutions with a physiological pH and CO2 (but little HCO3-), or pH and HCO3- (but little CO2). We have exploited these out-of-equilibrium solutions to introduce HCO3- exclusively to either the outside or inside of a squid giant axon, and verify the presence of a new K/HCO3 cotransporter. The out-of-equilibrium approach could be useful in a variety of applications for independently controlling CO2 and HCO3- concentrations and pH. © 2002 Nature Publishing Group.