This study investigates intracellular pH (pHi) changes in squid giant axons caused by CO₂, NH₃, and metabolic inhibitors. Using glass pH microelectrodes, researchers measured pHi in squid giant axons in artificial seawater (ASW) and observed significant changes when exposed to various substances. Exposure to 5% CO₂ caused a sharp decrease in pHi, followed by an overshoot upon removal of the gas. Prolonged exposure to CO₂ resulted in a slower rise in pHi during exposure and a more pronounced overshoot after removal. Similarly, exposure to NH₄Cl caused a rapid increase in pHi, followed by an undershoot upon return to ASW. Prolonged exposure to NH₄Cl led to a slow acidification during the plateau phase. Metabolic inhibitors caused pHi changes that were reversible depending on exposure duration. The study also found that the simultaneous passive movement of CO₂ and HCO₃⁻ could not explain the CO₂ results, suggesting the need for an active proton extrusion or sequestration mechanism. A mathematical model was developed to explain pH changes caused by NH₄Cl, considering the passive movement of both NH₃ and NH₄⁺. The model also explained the observed pH changes during CO₂ exposure, indicating the presence of an active proton extrusion mechanism. The study highlights the importance of considering both passive and active transport mechanisms in understanding pH changes in excitable tissues. The results suggest that intracellular pH regulation is complex and involves multiple factors, including the electrochemical gradient for H⁺ and the permeability of the cell membrane to various ions. The findings have implications for understanding pH regulation in other excitable tissues and the effects of metabolic inhibitors on cellular function.This study investigates intracellular pH (pHi) changes in squid giant axons caused by CO₂, NH₃, and metabolic inhibitors. Using glass pH microelectrodes, researchers measured pHi in squid giant axons in artificial seawater (ASW) and observed significant changes when exposed to various substances. Exposure to 5% CO₂ caused a sharp decrease in pHi, followed by an overshoot upon removal of the gas. Prolonged exposure to CO₂ resulted in a slower rise in pHi during exposure and a more pronounced overshoot after removal. Similarly, exposure to NH₄Cl caused a rapid increase in pHi, followed by an undershoot upon return to ASW. Prolonged exposure to NH₄Cl led to a slow acidification during the plateau phase. Metabolic inhibitors caused pHi changes that were reversible depending on exposure duration. The study also found that the simultaneous passive movement of CO₂ and HCO₃⁻ could not explain the CO₂ results, suggesting the need for an active proton extrusion or sequestration mechanism. A mathematical model was developed to explain pH changes caused by NH₄Cl, considering the passive movement of both NH₃ and NH₄⁺. The model also explained the observed pH changes during CO₂ exposure, indicating the presence of an active proton extrusion mechanism. The study highlights the importance of considering both passive and active transport mechanisms in understanding pH changes in excitable tissues. The results suggest that intracellular pH regulation is complex and involves multiple factors, including the electrochemical gradient for H⁺ and the permeability of the cell membrane to various ions. The findings have implications for understanding pH regulation in other excitable tissues and the effects of metabolic inhibitors on cellular function.