Two major forms of fumarate hydratase have been resolved in extracts prepared from a wide variety of mammalian cells by electrophoresis. Fractionation experiments with human and mouse cells suggest that one form (the slower migrating) is localized in the mitochondria, whereas the other form is predominant in the cytoplasm. Analysis of the segregation of the enzyme forms in human-mouse somatic cell hybrids indicates that a gene(s) necessary for the expression of both forms can be assigned to human chromosome 1(confirmation of a previous assignment by van Someren et al., 1974). Electrophoretic analysis suggests that the two forms may be interrelated. Furthermore, they both exhibit identical reactivity toward anti-fumarate hydratase antiserum. It is suggested that a modification of one form may occur in vivo and that the modification may be important in determining the intracellular localization of the enzyme.