This study investigates the cytoplasmic pH and free Mg²⁺ concentration in pig and mouse lymphocytes using four methods for pH measurement and three methods for Mg²⁺ assessment. The results show that the resting cytoplasmic pH is close to 7.0, and the free Mg²⁺ concentration is around 1 mM. The digitonin null-point technique, freeze-thawed cell pellet measurements, a newly synthesized intracellularly trappable fluorescent pH indicator, and ³¹P nuclear magnetic resonance (NMR) were used to measure pH. The A23187 null-point method, Mg-sensitive electrode measurements, and ³¹P NMR were used to assess free Mg²⁺. The study found that mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. The results highlight the importance of maintaining steady-state cytoplasmic pH and free Mg²⁺ levels for various cellular processes.This study investigates the cytoplasmic pH and free Mg²⁺ concentration in pig and mouse lymphocytes using four methods for pH measurement and three methods for Mg²⁺ assessment. The results show that the resting cytoplasmic pH is close to 7.0, and the free Mg²⁺ concentration is around 1 mM. The digitonin null-point technique, freeze-thawed cell pellet measurements, a newly synthesized intracellularly trappable fluorescent pH indicator, and ³¹P nuclear magnetic resonance (NMR) were used to measure pH. The A23187 null-point method, Mg-sensitive electrode measurements, and ³¹P NMR were used to assess free Mg²⁺. The study found that mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. The results highlight the importance of maintaining steady-state cytoplasmic pH and free Mg²⁺ levels for various cellular processes.