A human-rat hybrid form of enolase (beta gamma) was prepared by dissociation and reassociation of a mixture of human beta beta and rat gamma gamma enolases, followed by isolation of the hybrid form from the parental homodimeric enolases with DEAE-Sephadex column chromatography. The human-rat beta gamma enolase had a specific activity similar to those of human beta beta and rat gamma gamma enolases. The optimal pH, stability against heating, and Km for 2-phosphoglycerate of the hybrid enolase were also similar to those of the homodimeric enolases.