Arylsulfatase-C and sterol sulfatase were thought to be identical enzymes whose X-linked locus escapes inactivation. However, recent evidence shows that they are not identical but that arylsulfatase-C in human fibroblasts exists in two isozymic forms, designated as slow and fast. We now report that the two forms are enzymatically different. When assayed with an artificial fluorogenic substrate, the slow form showed a pH optimum of 8.00 and a Km of 228 microM. In contrast, the fast form showed a pH optimum of 7.67 and a Km of 86.7 microM with substrate inhibition occurring above 0.33 mM. The heat stability of the fast form was slightly below that of the slow form. Polyclonal antibodies raised against the slow form did not cross-react with the fast form. Hence, the two isozymic forms of arylsulfatase-C are enzymatically and structurally different and the slow form is associated with sterol sulfatase activity.