beta-N-Acetylhexosaminidase (hexosaminidase) I, which has an intermediate charge character between those of hexosaminidases A(alpha beta 2) and B[beta beta)2), was purified 1,500-fold from human placenta by procedures including chromatographies on concanavalin A (Con A)-Sepharose and an immunoadsorbent column. The isolated hexosaminidase I was heat-stable, and antigenically cross-reactive to anti-beta chain-IgG but not to anti-alpha chain-IgG. The results of substrate specificity experiments using 3H-labeled natural substrates indicated that the hexosaminidase I hydrolyzed Gb4Cer to Gb3Cer but not GM2 to GM3. The tryptic peptide map of the hexosaminidase I was similar to that of hexosaminidase B, though some differences were observed. The hexosaminidase I after treatment with neuraminidase or endo-beta-N-acetylglucosaminidase H was partly converted to less acidic forms. Treatment of the hexosaminidase I with acid phosphatase did not change the charge character. Therefore hexosaminidase I is an acidic variant form of hexosaminidase B, possibly resulting from sialylation and the presence of phosphodiester bonds at the carbohydrate moiety.