A new, rapid, and sensitive method for assaying phenol sulfotransferase activity toward 2-naphthol is described. The product 2-naphthyl sulfate is quantitated fluorometrically. Optimal wavelengths for excitation and emission were determined by recording the three-dimensional fluorescence spectra of the substrate and the product. The new method is applicable to crude cell or tissue homogenates as well as to further purified preparations. A comparison to another widely used method is given to point out the advantages provided by the new procedure. In particular, sensitivity and accuracy of both methods are evaluated and the influence of interfering substances on both systems is compared. These results clearly indicate the superiority of the new method.