Many enzymes of animals, especially enzymes that regulate metabolic pathways, exhibit one or more types of inter- and intracellular compartmentation. A given type of catalytic activity is effected by different isoforms (isozymes) of a class of enzyme in different tissues, cells, or organelles, and many enzymes alter their locations within the cell under different physiological conditions. These compartmentation properties reflect major features of animal organization and function, e.g., the occurrence of different metabolic tasks in different tissues or cellular compartments, and enormous changes in muscle metabolic rate during intense locomotion. Enzymes of glycolysis, notably phosphofructokinase (PFK) and hexokinase (HK), manifest these forms of compartmentation. PFK occurs in several isozyme forms that differ in kinetic properties. Skeletal muscle PFK binds reversibly to actin in a pH-dependent manner. Binding of PFK to actin affects the enzyme's catalytic activity, sensitivity to regulatory modulators, and structural stability in the face of decreasing intracellular pH ([pH.sub.i]). Eucaryotic PFKs arose from a gene duplication/fusion event of an ancestral procaryotic PFK. This increase in subunit mass from 36 kDa to 82 kDa in animal PFKs facilitated the evolution of additional regulatory sites and, we hypothesize, an actin binding capacity which varies among PFK isoforms. In vertebrates, hexokinase occurs in several isoforms that differ in kinetic properties and ability to bind to the outer surface of the mitochondrion. Binding of HK to mitochondria plays an important role in regulating glucose metabolism in glucose-dependent tissues like brain. The different abilities of vertebrate HK isozymes to bind to mitochondria are due to charge differences on the isozymes' surfaces. Invertebrate HKs are smaller than vertebrate HKs (ca. 50 kDa vs. 100 kDa), and cannot bind to mitochondria. The evolution of multiple isozyme forms of animal enzymes, and the evolution of capacities for reversible intracellular compartmentation through increases in protein size, are discussed in the context of the large genome sizes characteristic of most animals.