The abundant secreted protein with molecular weight of 32,000 was purified from the culture medium of suspension-cultured pumpkin (Cucurbita sp.) cells. Two steps, ammonium sulfate fractionation and Sepharose 6B column chromatography, were sufficient for purification to homogeneity. Antibodies against the pure protein were used to show that a protein of the same size is made by callus cells. There is considerable homology between the amino-terminal amino acid sequence of this secreted protein and chitinase isolated from tobacco (Nicotiana tabacum L.) or bean (Phaseolus vulgaris L.).Much attention has been focused recently on pathogenesisrelated proteins that have been implicated in the defense reaction of plants against pathogens including viruses (3, 26), bacteria (1), viroids (6), and fungi (12). These proteins have been found in many plant species since they were first detected in tobacco reacting hypersensitively to tobacco mosaic virus (29). These proteins are induced as a response to stress (23), infection (31), and chemicals (31, 32). Furthermore, in cultured tobacco cells, they are also induced by combinations of the plant hormones auxin and cytokinin and are secreted into the culture medium (2, 24). In tobacco, 10 major pathogenesis-related proteins can be detected (30). However, their biological function has largely remained elusive. Recently, three of them were shown to have 3-1,3-glucanase activity (15, 17), and four proteins have been identified as chitinases ( 17,19,22); it may catalyze the hydrolysis ofthe d-1,4 linkages of N-acetyl-D-glucosamine polymer chitin present in the cell walls of many fungi.When the culture medium in pumpkin cell suspension cultures was analyzed by SDS-PAGE, a major polypeptide band was observed at the position corresponding to Mr of 32,000 (10). In the present study, we have purified the abundant secreted protein (SP32) and prepared an antiserum against it. We also determined the amino acid composition