Temperature-induced pH changes in white muscle tissue of three eelpout populations with different levels of eurythermy (the cold stenothermal Antarctic species Pachycara brachycephalum and the temperate eelpout Zoarces viviparus from the North Sea and the Baltic Sea) were monitored online by use of in vivo 31P-NMR in unrestrained, unanaesthetized fish. An intracellular pH (pHi) change of around -0.015pH units/°C was observed in all eelpout populations in accordance with the α-stat hypothesis. The pH change was completed earlier (within 4h) in the stenothermal Antarctic eelpout than in the Baltic population (within 8 h) and latest (not within 12 h) in the eurythermal North Sea population. These findings confirm the hypothesis that the kinetics of temperature-dependent pHi regulation is reflected by the relative contribution of active and passive processes to a temperature-induced pH change. The extent of passively induced pHi changes is in line with the general hypothesis that the temperature-dependent adjustment of pHi occurs mostly by active mechanisms in eurythermal animals, whereas in stenothermal animals pH changes are largely elicited by passive processes. Temperature changes had no influence on high-energy phosphates like phosphocreatine and ATP or on the Gibbs free energy change of ATP hydrolysis (ΔG/Δζ). © 2003 Elsevier Science Ltd. All rights reserved.