Using colloidal [3H] chitin as a substrate, we provide the first demonstration of a chitinase in human leukocytes; chitinolytic activity in whole and disrupted leukocyte preparations (approximately 0.6 and 5.5 nmol of N-acetylglucosamine [GlcNAc] released min-1 mg of protein-1, respectively) was partially inhibited by the specific chitinase inhibitor allosamidin (9 microM). Following fractionation of the leukocytes, much higher levels of chitinase activity were detected in granulocyte-rich homogenates (approximately 7.2 nmol of GlcNAc released min-1 mg of protein-1) than in lymphocyte- and monocyte-rich homogenates (approximately 0.22 and 0.26 nmol of GlcNAc released min-1 mg of protein-1, respectively). Low levels of chitinase activity were detected in human serum (approximately 4 pmol of GlcNAc released min-1 mg of protein-1). Chitinolytic activity in granulocyte-rich homogenates and serum was partially inhibited by allosamidin (9 microM). Proteins with chitinolytic activities (approximate molecular masses, 48 and 56 kDa) distinct from lysozyme (14.3 kDa) were detected on polyacrylamide gels following the electrophoresis of human granulocyte-rich preparations. Chitinase activity, detected consistently in serum and leukocytes from all human volunteers investigated, may contribute to the protection of the host by cleaving chitin in the cell walls of fungal pathogens.