Abstract 1. 1. A third exo- β- N-acetylglucosaminidase (III) was purified from pupal hemolymph of the tobacco hornworm, Manduca sexta by ammonium sulfate fractionation, anion-exchange chromatography, hydroxylapatite chromatography and gel filtration. 2. 2. Two forms of the enzyme were separated by polyacrylamide gel electrophoresis, one composed of a single polypeptide chain with MW app = 6.1 × 10 4 and the other composed of two chains with MW app = 5.3 × 10 4 and 1 × 10 4. 3. 3. The K m and k cat values for pNpβGlcNAc were265 μm and 374 sec −1, respectively. 4. 4. The pH rate profile suggested that two groups with pK a = 8.1 and 3.7 participate in catalysis. 5. 5. N-Acetylglucosamine and 1-O-methyl derivatives inhibited III in a competitive fashion with K i values = 3–10 mM. 6. 6. III was related immunologically to exo- β- N-acetylglucosaminidases I and II which are also present in pupal hemolymph. 7. 7. Only I was detected in pharate pupal molting fluid and integument.