We assessed hypoxia tolerance in 11 species of fish from the superfamily Cottoidea (commonly called sculpins) that are known to differ in their critical O₂ tensions (P_crit) and examined whether hypoxia tolerance correlated with larger substrate stores and higher maximal activity of enzymes associated with anaerobic adenosine triphosphate production (especially glycolysis). Among the sculpins studied, there was large variation in time to loss of equilibrium (LOE₅₀) at torr, with values ranging between 25 and 538 min, and the variation in LOE₅₀ was correlated with P_crit. Our measures of time to LOE₅₀ and P_crit were regressed against maximal enzyme activities of lactate dehydrogenase (LDH), pyruvate kinase (PK), creatine phosphokinase (CPK), and citrate synthase (CS) as well as the concentrations of glycogen, glucose, and creatine phosphate in the brain, liver, and white muscle. In the brain, there was a phylogenetically independent relationship between P_crit and tissue LDH, PK, CPK, and CS activities expressed relative to tissue mass. Hypoxia-tolerant sculpins (those with low P_crit values) had higher levels of brain LDH, PK, CPK, and CS than did hypoxia-sensitive sculpins. Similarly, LOE₅₀ regressed against brain LDH, PK, and CPK activities expressed relative to tissue mass, with the more hypoxia-tolerant species (i.e., those with higher LOE₅₀) having higher enzyme activities. However, when the phylogenetic relationship among our sculpins was taken into account, only the relationship between hypoxia tolerance and LDH activity remained significant. When enzyme activities were expressed relative to total soluble protein in the tissue, the only relationships that remained were between brain LDH activity and P_crit and LOE₅₀. In liver and white muscle, there were no relationships between the measures of hypoxia tolerance and enzyme activity or metabolite content. Overall, our analysis suggests that hypoxia-tolerant sculpins maintain higher maximal activities of some of the enzymes involved in anaerobic metabolism in the brain, and this may be an adaptation to hypoxia.