A new, simple and sensitive method is described for assay of serum gamma-glutamyltransferase activity based on the hydrolysis of the substrate L-gamma-glutamyl-3-carboxy-4-nitranilide which offers the advantage of producing directly its own chromogen. The substrate is highly soluble and the method can therefore easily be adapted to any equipment for the automated assay of gamma-glutamyltransferase. The activities at measured optimum substrate concentration are slightly higher than with the Szasz ((1969), Clin. Chem. 15, 124-136) method in which L-glutamyl-p-nitroanilide is used as substrate.