A new enzyme-linked immunoassay for the determination of anti-erythrocyte antibodies is described. A fluorogenic substrate of alkaline phosphatase, the 4-MUP, was used to reveal the antigen-antibody reaction. This substrate was chosen in order to avoid interferences with haemoglobin, since it emits a light with a wavelength outside the absorption spectrum of haemoglobin. This method reaches a sensitivity in the order of 1 ng/ml. Sera from 66 Rh-immunized pregnant women and 20 control subjects have been studied. The specificity and reproducibility of the results and their good correlation with the biological and clinical parameters of the hemolytic disease of the newborn confirm the validity of the method, encouraging its routine use.