Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, β-glucuronidase and β-N- acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1–1.5 units lower when electrofocusing was done in a pH 3–10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as a single, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3–5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1% p- nitrophenyloxamic acid, 0.1 M glycine buffer, pH 9) and including 0.1% p- nitrophenyloxamic acid, an inhibitor of lysosomal neuraminidase and cathepsin D, in the pH gradient, arylsulfatase, β-glucuronidase and β-N- acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) at 0–4 °C and omitting p- nitrophenyloxamic acid from the gradient, the acidic form of β-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and β-N- acetylhexosaminidase were increased 0.6–1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and β-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and β-glucuronidase were largely lost, the isoelectric point of the acidic form of β-N- acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5–1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 produced similar, though less marked, effects. The loss of the acidic forms of the hydrolases during these various treatments was due to their conversion to more basic forms. These findings confirm the glycoprotein nature of these lysosomal hydrolases and show that N- acetylneuraminic acid is the major source of anionic groups in both the acidic and basic forms of these enzymes.