The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyru-vate kinase were studied in breast cancer tissues, in comparison to benign breast disease and normal breast tissues. The enzyme activities in breast cancer were significantly increased compared to normal and benign breast tissues (p < 0.001). Also the increase in activity in benign disease compared to normal was statistically significant (p < 0.001). Within the group of benign diseases, fibroadenomas could be distinguished from fibrocystic disease, the former generally showing higher activities compared to the latter (p ≤ 0.05). Carcinoma subgroups, classified according to their histology, could not be recognized enzymologically. In addition, isozyme composition of pyruvate kinase and enolase was studied. We did not find a significant shift towards K type pyruvate kinase expression in benign disease compared to normal breast tissues. Also fibroadenomas did not differ from fibrocystic disease. However, the amount of K type pyruvate kinase in carcinomas proved to be significantly higher in comparison to benign disease and normal breast tissues (p < 0.001). Expression of αγ-enolase in normal breast tissue was virtually absent. In benign disease only a minority of specimens did show the hybrid αγ-enolase. Nearly all carcinomas had αγ-enolase expression and in 20% of the carcinomas γγ-enolase could be detected (so-called neuron-specific enolase). By discriminant analysis, the function giving the best discrimination compared to the histological data was based on natural logarithm aldolase and the total of γ-enolase subunits. Contrary to expectation, the regulator enzymes of glycolysis; i.e., hexokinase, phosphofructokinase and pyruvate kinase were not included in this discriminant function. The best fit produced a 90% correct classification in both benign and malignant disease. If these findings are confirmed to a larger series, the discrimination is sufficiently strong to form the basis of a clinically useful tool.