Enolase isozyme composition was studied using both electrophoretic and chromatographic methods in rat medullary thyroid carcinomas (MTC), differing in their degree of differentiation. In well-differentiated rat tumors (DMTC), both the alpha- and gamma-subunits of enolase were expressed, resulting in alpha alpha, alpha gamma, and gamma gamma isozymes. The relatively high amount of alpha gamma and gamma gamma isozymes (neuron-specific enolase [NSE] ) was indicative of the presumed neuroectodermal origin of these tumors. In contrast, highly undifferentiated or anaplastic tumors (AMTC) were characterized by a decrease in expression of the gamma-subunit. Hence, the majority of enolase isozymes were alpha alpha dimers, with only a few percent alpha gamma hybrids remaining. These shifts from neuron-specific to non-neuronal isozymes in rat MTC were compared with human MTC and discussed with respect to neuronal differentiation and the clinical significance of NSE measurements in serum as a marker for amine precursor uptake and decarboxylation cell-derived neoplasms.