A unique electrophoretic form of carbonic anhydrase is characteristic of some laboratory-maintained mice of the wild mouse species Mus spretus. This isozyme has been characterized by cellulose acetate electrophoresis and by isoelectric focusing. It is proposed that this isozyme be called CAR-2C and that its encoding allele be designated Car-2c. Fertile hybrids of Mus spretus and C57BL/6J (Car-2a) show both CAR-2A and CAR-2C bands of approximately equal intensity. The CAR-2C isozyme is readily identified by electrophoresis on 75-mm cellulose acetate strips because it migrates significantly faster than the isozymes of inbred mice, the CAR-2A and CAR-2B that do not separate from one another under standard conditions. Isoelectric focusing cleanly resolves all three of these CAR-2 forms. Mus hortulanus, although closely related to Mus spretus in other biochemical-genetic characteristics, has a CAR-2-homologous isozyme that is distinctly different from the CAR-2C of Mus spretus and from the isozymes of the common inbred strains.