Tubulins purified from brain tissue of Antarctic fishes assemble in vitro to form microtubules at the low temperatures experienced by these extreme psychrophiles (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). We have initiated studies to determine the structural requirements for assembly of Antarctic fish tubulins at low temperatures. As a first step we have compared the heterogeneity, structures, amino acid compositions, and net charge of brain tubulins purified from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from the temperate channel catfish (Ictalurus punctatus), and from a mammal (the cow). Each preparation contained the alpha- and beta-tubulins and was free of microtubule-associated proteins. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into approximately 20 isoelectric variants. The distributions of the isotubulins from the cold-adapted fishes were similar but differed significantly from those of tubulins from catfish and cow. The average isoelectric points of the alpha- and beta-tubulins from the Antarctic fishes were more basic than the isoelectric points of the corresponding tubulins from bovine brain. Peptide mapping confirmed that tubulins from the Antarctic fishes and the mammal differed in structure. The amino acid compositions of fish and mammalian tubulins were similar, but Antarctic fish tubulins apparently contained fewer Glx residues than did catfish or bovine tubulins. Finally, native tubulins from an Antarctic fish and the cow differed slightly in net negative charge. Thus, brain tubulins from the cold-adapted fishes differ structurally from the tubulins of a temperate fish and of a mammal.