A direct and continuous kinetic method for the fluorimetric assay of various hydrolases by using new, highly water-soluble substrates is described. The latter consist of esters of strongly fluorescent 1-hydroxypyren-3,6,8-trisulfonic acid trisodium salt with acetic, butyric, caprylic, and oleic acid. Km and vmax values are given for the hydrolytic activity of porcine liver carboxylic ester hydrolase, wheat germ lipase, candida cylindracea lipase, hog kidney acylase I, and bovine pancreas alpha-chymotrypsin, while others (acetylesterase, trypsin, and cholinesterase) were studied qualitatively. By proper choice of the substrate, a fair selectivity may be achieved. Detection limits as low as 1 microgram enzyme/ml are found in some cases. Advantages of these new substrates over existing ones are briefly discussed.