The study measures cytoplasmic pH (pHi) and free Mg²+ concentration in pig and mouse lymphocytes. pH was measured using four methods: digitonin null-point, freeze-thawed cell pellet pH, 31P NMR of inorganic phosphate, and a fluorescent pH indicator. In HEPES-buffered saline at 37°C, pH was close to 7.0. Addition of physiological levels of HCO3⁻ and CO2 transiently acidified the cells by ~0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. Free Mg²+ was measured using three methods: external Mg²+ null-point, Mg-sensitive electrodes, and 31P NMR of ATP γ-phosphate. Total cell Mg²+ was ~12 mmol per liter cell water, with intracellular Mg²+ estimated at ~0.5 mM. The null-point method gave ~0.9 nM, while electrode measurements gave 1.35 mM, thought to be an overestimate. Exposure to Con A for 1 hour had no detectable effect on total or free Mg²+. Cytoplasmic pH and free Mg²+ are critical for many intracellular processes. Understanding these parameters is essential for studying cellular mechanisms. The study used various methods to measure pH and Mg²+ in lymphocytes, including digitonin null-point, freeze-thawed cell pellet pH, and fluorescent indicators. The results showed that pH was around 7.0 and free Mg²+ was ~1 mM. The study also found that mitogenic doses of Con A had no measurable effect on pH or free Mg²+ within the first hour. The results suggest that pH and Mg²+ are important for cell activation, but their exact roles require further investigation. The study highlights the importance of accurate measurements of pH and Mg²+ in understanding cellular processes.The study measures cytoplasmic pH (pHi) and free Mg²+ concentration in pig and mouse lymphocytes. pH was measured using four methods: digitonin null-point, freeze-thawed cell pellet pH, 31P NMR of inorganic phosphate, and a fluorescent pH indicator. In HEPES-buffered saline at 37°C, pH was close to 7.0. Addition of physiological levels of HCO3⁻ and CO2 transiently acidified the cells by ~0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. Free Mg²+ was measured using three methods: external Mg²+ null-point, Mg-sensitive electrodes, and 31P NMR of ATP γ-phosphate. Total cell Mg²+ was ~12 mmol per liter cell water, with intracellular Mg²+ estimated at ~0.5 mM. The null-point method gave ~0.9 nM, while electrode measurements gave 1.35 mM, thought to be an overestimate. Exposure to Con A for 1 hour had no detectable effect on total or free Mg²+. Cytoplasmic pH and free Mg²+ are critical for many intracellular processes. Understanding these parameters is essential for studying cellular mechanisms. The study used various methods to measure pH and Mg²+ in lymphocytes, including digitonin null-point, freeze-thawed cell pellet pH, and fluorescent indicators. The results showed that pH was around 7.0 and free Mg²+ was ~1 mM. The study also found that mitogenic doses of Con A had no measurable effect on pH or free Mg²+ within the first hour. The results suggest that pH and Mg²+ are important for cell activation, but their exact roles require further investigation. The study highlights the importance of accurate measurements of pH and Mg²+ in understanding cellular processes.