Summary1Four major enolase isozymes have been identified in human tissues and are referred to as L, M, ‘intermediate’ and ‘fast’. The M isozyme is the major form found in skeletal muscle and heart extracts and the L isozyme the major form found in extracts of liver and most other tissues The ‘intermediate’ and ‘ fast’ isozymes are most active in brain but are observed as weak components in most other tissues including heart but are not seen in skeletal muscle. It was observed that during fetal development of heart and skeletal muscle the L form declines in activity while the M form increases in activity.2The kinetic properties, heat stabilities and molecular sizes of the main enolase isozymes have been compared. Although the isozymes share many features in common, the ‘fast’ isozyme is more stable when subjected to heat treatment than either the L or M isozymes. Further, the ‘fast’ isozyme retains its dimeric structure and activity in the absence of magnesium ions while the L and M isozymes dissociate and lose activity. The ‘intermediate’ isozyme has properties which are intermediate to those of the L and ‘fast’ isozymes.3The ‘intermediate’ isozyme can be partially dissociated to equal quantities of L and ‘fast’ isozymes by storage at room temperature or by freezing and thawing in the presence of 2 m‐NaCl. Conversely, mixtures of L with ‘fast’ and M with ‘fast’ give rise to an ‘intermediate’ isozyme after freezing and thawing.4Evidence derived from this study has led to the suggestion that three separate gene loci are involved in the determination of human enolase. It is proposed that one of these,ENO1, determines the L isozyme which is the homodimer αα;another locus,ENO2, determines the ‘fast’ isozyme which is the homodimer ββ; and the third locus,ENO3, determines the M isozyme which is the homodimer γγ. The ‘intermediate’ isozyme seen as a strong component in brain and as a weak component in most other tissues is thought