Two isozymes of creatine kinase have been purified differentially from mitochondrial and cytoplasmic subfractions of intestinal epithelial cells. These intestinal epithelial cell creatine kinases were indistinguishable from the cytoplasmic (B-CK) and mitochondrial (Mi-CK) creatine kinase isozymes of brain when compared by SDS-PAGE, cellulose polyacetate electrophoresis, and peptide mapping. In intestinal epithelial cells, immunolocalization of the Mi-CK isozyme indicates that it is associated with long, thin mitochondria, which are excluded from the brush border at the apical end of each cell. In contrast, immunolocalization of the B-CK isozyme indicates that it is concentrated distinctly in the brush border terminal web domain. Although absent from the microvilli, B-CK also is distributed diffusely throughout the cytoplasm. Terminal web localization of B-CK was maintained in glycerol-permeabilized cells and in isolated brush borders, indicating that B-CK binds to the brush border structure. The abundance and localization of the mitochondrial and cytoplasmic creatine kinase isozymes suggest that they are part of a system that temporally and/or spatially buffers dynamic energy requirements of intestinal epithelial cells.