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Enolase, a glycolytic enzyme, is a multifunctional protein with location diversity. We revealed the intracellular distribution of enolase isozymes, such as alphaalpha-, alphagamma- and gammagamma-enolases, in rat brain synaptic terminals by biochemical and immunoelectron microscopic analyses. Specific activity of enolase of synaptic plasma membrane fraction (SPM2) obtained from synaptosomes was 23.2 +/- 4.4 x 10(-2) micromol/mg protein/min in the presence of 0.25% Triton X-100 and that of synaptosomal cytoplasm (LS) was 67.4 +/- 12.1 x 10(-2) micromol/mg protein/min. About half of enolase activity in synaptosomes was distributed to soluble fraction while the remaining stayed in particulate membrane fractions by ultracentrifugation. Immunoblot analysis of the fractions demonstrated both alpha and gamma subunits were distributed in SPM. In addition, immunoelectron microscopic analysis also revealed that both subunits were immunoreactive on the SPM. Using coimmunoprecipitation assay, we confirmed that the enolase was present not only as a homodimer form but also as an alphagamma hybrid form associated with membrane, where both subunits were coimmunoprecipitated from lysate of SPM2 in the presence of Mg(2+). These findings indicate that all forms (alphaalpha, alphagamma, and gammagamma) of enolase translocate to the plasma membrane and associate with some components in the SPM.