The beta-hexosaminidase isozymes are produced through the combination of alpha and beta subunits to form any one of three active dimers (monomeric subunits are not functional). Heterodimeric hexosaminidase A (alpha beta) is the only isozyme that can hydrolyze GM2 ganglioside in vivo, requiring the presence of the GM2 activator protein. Hexosaminidase S (alpha alpha) exists but is not considered a physiological isozyme. Although hexosaminidase B (beta beta) is present in normal human tissues, it has no known unique function in vivo. However, a unique function for the beta-active site present in both hexosaminidase A and B has been indicated in a previous study of the various substrate specificities of the homodimeric forms of hexosaminidase (S and B). It was concluded that the alpha-active site is only able to efficiently hydrolyze negatively charged substrates, and the beta-active site is only able to hydrolyze neutral substrates. When this model of nonoverlapping alpha- and beta-substrates is extrapolated to heterodimeric hexosaminidase A, it has a major effect on the interpretation of recent results relating to the mode of action of the GM2 activator protein. In this report, we directly examine these substrate specificities using a novel form of hexosaminidase A containing an inactive beta subunit, produced in permanently transfected CHO cells. We demonstrate that, whereas the beta-active site has the same substrate specificities in either its A-heterodimeric or B-homodimeric forms, the alpha-active site in the A-heterodimer has different kinetic parameters than the alpha-active site in the S-homodimer. We conclude that the alpha and beta subunits in hexosaminidase A participate equally in the hydrolysis of neutral substrates.