In order to detect differences between various multiple forms of gamma-glutamyltransferase, the activation energy was measured. In the serum of patients with liver diseases, activation energy was measured. In the serum of patients with liver diseases, activation energy of the serum enzyme is higher than in normal individuals (41.9 +/- 1.2 vs. 38.9 +/- 1.5 kJ/mol, p less than 0.05). Neuraminidase treatment resulted in a reduction of activation energy. Various multiple forms of serum gamma-glutamyltransferase, as prepared by lectin affinity chromatography (concanavalin A, Ricinus communis I and II, wheat germ agglutinin) showed activation energy differences between binding and nonbinding fractions. Similar results were observed in seminal plasma gamma-glutamyltransferase, when patients with accessory gland infection were compared with a reference population. Our results suggest that the activation energy depends upon differences in the carbohydrate part of the enzyme. The low gamma-glutamyltransferase activation energy of tissue extracts increased significantly after butanol extraction and was then comparable with serum activation energy values, which suggests that lipid-binding is a factor in activation energy variation. In most cases, gamma-glutamyltransferase activities measured at a certain temperature can be easily converted to a corresponding activity at another temperature, but in severe liver disease significant errors may be introduced when simple temperature conversion factors are used.