The trisaccharide 6-sulfo-N-acetylgalactosamine-glucuronic acid-6-sulfo-N-acetyl-[1-3H]galactosaminitol was used as a substrate for the determination of N-acetylgalactosamine-6-sulfate sulfatase activity. The amount of liberated sulfate was measured indirectly by separating monosulfated reaction products from the substrate on Dowex 1 X 2 microcolumns in a simple two step procedure. Fibroblast homogenates from patients with various genotypes, except classical Morquio's disease, released 410 +/- 90 pmol sulfate/h/mg cell protein. The enzyme exhibited a pH optimum of pH 4.8 and a KM of about 1 X 10(-4) mol/1. It was strongly inhibited by phosphate, sulfate and chloride ions. In three cell lines from patients with classical Morquio's disease a residual activity between 1 and 2% of the mean normal activity was found. All cell lines tested released sulfate from 6-sulfo-N-acetylglucosamine-glucuronic acid-[1-3H]-anhydromannitol. Cell extracts from cultured amniotic fluid cells exhibited a N-acetylgalactosamine-6-sulfate sulfatase activity between 120 and 320 pmol/h/mg protein. An enzyme activity of 370 +/- 100 pmol sulfate/h/mg protein was found in peripheral leucocytes from healthy donors. The determination of N-acetyl-galactosamine-6-sulfate sulfatase activity in one family with an affected patient indicated that the enzyme deficiency is also expressed in leucocytes.