Hybridization studies have been carried out in vitro using mixtures of partially purified isoenzymes 1 and 3 of rat enolase (2-phospho-D-glycerate hydrolyase, EC 4.1.1.11). Immunological methods were used to demonstrate the formation of the hybrid. 2. Immunological analysis of the elution peaks from QAE-Sephadex chromatography of heart enolase indicates the occurrence in vivo of the hybrid enolase 2. 3. Developmental changes in the proportions of isoenzymes 1, 2 and 3 in heart and skeletal muscle of rat have been studied quantitatively. In both tissues isoenzyme 1 predominates in the foetus, but is partially replaced by 2 and 3 in adult heart and completely by 3 in the adult muscle. 4. Evidence is given of the binomial distribution of the proportions of the three isoenzymes in the developing heart. 5. Phylogenetic studies of the immunological properties of enolases from muscle, liver and heart have been carried out. 6. It is concluded that the three isoenzymes arise from two independent genetic loci and it is suggested that these evolved from a common ancestral gene 200-300 million years ago.
