The alpha- and beta-subunits of beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) are synthesized in the rough endoplasmic reticulum as prepropolypeptides. After the loss of the signal peptide and formation of enzymatically active dimers, the pro-isoenzymes are transported through the Golgi and into the lysosome for proteolytic and glycolytic processing to their stable mature forms. Maturation includes the hydrolysis, and previously presumed loss, of small N-terminal peptides from each propolypeptide. A recent report characterizing the processing of the beta-prepropolypeptide in beta-hexosaminidase from a human fibroblast cell line [(1989) J. Biol. Chem. 264, 3380-3384] reported that the small pro-beta peptide was retained through a disulfide bond in the mature subunit, and that it was glycosylated. We have confirmed this result in normal human tissue. However, we report a different N-terminal for the mature pro-beta peptide. Furthermore, we have found that the pro-alpha peptide is similarly retained in the mature alpha-subunit through its single cysteine residue and that each pro-peptide undergoes C-terminal processing.