Disruption of neuronal function is likely to influence limits to thermal tolerance. We hypothesized that with acute warming the structure and function of neuronal membranes in the Antarctic notothenioid fish Chaenocephalus aceratus are more vulnerable to perturbation than membranes in the more thermotolerant notothenioid Notothenia coriiceps. Fluidity was quantified in synaptic membranes, mitochondrial membranes, and myelin from brains of both species of Antarctic fishes. Polar lipid compositions and cholesterol contents were analyzed in myelin; cholesterol was measured in synaptic membranes. Thermal profiles were determined for activities of two membrane-associated proteins, acetylcholinesterase (AChE) and Na+/K+-ATPase (NKA), from brains of animals maintained at ambient temperature or exposed to their critical thermal maxima (CTMAX). Synaptic membranes of C. aceratus were consistently more fluid than those of N. coriiceps (P < 0.0001). Although the fluidities of both myelin and mitochondrial membranes were similar among species, sensitivity of myelin fluidity to in vitro warming was greater in N. coriiceps than in C. aceratus (P < 0.001), which can be explained by lower cholesterol contents in myelin of N. coriiceps (P < 0.05). Activities of both enzymes, AChE and NKA, declined upon CTMAX exposure in C. aceratus, but not in N. coriiceps. We suggest that hyper-fluidization of synaptic membranes with warming in C. aceratus may explain the greater stenothermy in this species, and that thermal limits in notothenioids are more likely to be influenced by perturbations in synaptic membranes than in other membranes of the nervous system.