Genetics of human lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolase, EC 3.1.6.1), associated with childhood disease, has been studied with human-rodent somatic cell hybrids. Deficiency of arylsulfatase A (ARS A ) in humans results in a progressive neurodegenerative disease, metachromatic leukodystrophy. Deficiency of arylsulfatase B (ARS B ) is associated with skeletal and growth malformations, termed the Maroteaux-Lamy syndrome. Simultaneous deficiency of both enzymes is associated with the multiple sulfatase deficiency disease, suggesting a common relationship for ARS A and ARS B . The genetic and structural relationships of human ARS A and ARS B have been determined by the use of human-Chinese hamster somatic cell hybrids. Independent enzyme segregation in cell hybrids demonstrated different chromosome assignments for the structural genes, ARS A and ARS B , coding for the two lysosomal enzymes. ARS A activity showed concordant segregation with mitochondrial aconitase encoded by a gene assigned to chromosome 22. ARS B segregated with β-hexosaminidase B encoded by a gene assigned to chromosome 5. These assignments were confirmed by chromosome analyses. The subunit structures of ARS A and ARS B were determined by their electrophoretic patterns in cell hybrids; a dimeric structure was demonstrated for ARS A and a monomeric structure for ARS B . Although the multiple sulfatase deficiency disorder suggests a shared relationship between ARS A and ARS B , independent segregation of these enzymes in cell hybrids did not support a common polypeptide subunit or structural gene assignment. The evidence demonstrates the assignment of ARS A to chromosome 22 and ARS B to chromosome 5. A third gene that affects ARS A and ARS B activity is suggested by the multiple sulfatase deficiency disorder.