Phomopsis cucurbitae is a latent infection fungus which causes muskmelon fruit decay only after harvest. It can cause severe losses during fruit storage and marketing in the U.S.A., Japan and some Central American countries. Previous studies showed that P. cucurbitae produced polygalacturonase (PG) in both culture and muskmelon fruit tissue. In this study, a prominent PG isozyme (PG1) produced by P. cucurbitae in decayed fruit was purified to homogeneity by a procedure of extraction, ultrafiltration, preparative isoelectric focusing, anion exchange, and gel filtration chromatography. The isozyme had a primary endo-activity, a relative molecular weight of 54 kD according to SDS-PAGE, and a pI of 4·2 based on IEF-PAGE. The isozyme was active in a low pH range from 2 to 6, and was optimum at pH 5. Optimum temperature for the activity of PG1 was 40–45°C. The Km and Vmax on polygalacturonic acid were 44·7 μg ml−1 and 0·14 U μg−1 enzyme, respectively. The purified PG1 effectively macerated mature fruit tissue, suggesting that the isozyme may be involved in the pathogenesis of Phomopsis fruit rot of muskmelon.