The identification of a second structural gene mutation at the feline arylsulfatase B locus (MPS VIb) provided the opportunity to investigate the expression of allelism by characterization of the residual enzymatic activity in feline mucopolysaccharidosis VI, an animal analogue of human Maroteaux-Lamy syndrome. Matings were designed to produce affected homozygotes who were homoallelic for the MPS VIa and MPS VIb mutations or heteroallelic genetic compounds (MPS VIa/VIb). The physicokinetic and immunological properties of the partially purified residual hepatic arylsulfatase B isozymes from the affected homoallelic and heteroallelic cats were compared to those of the normal feline enzyme. The residual hepatic arylsulfatase B activities from the inbred MPS VIa and MPS VIb homozygotes were distinguished by differences in physicokinetic and immunological properties. The newly identified mutant isozyme b had abnormal kinetic properties toward artificial and natural substrates, normal cryo- and thermostabilities, a normal molecular weight and an altered electrophoretic mobility. Polyacrylamide gel electrophoresis demonstrated that the mutant b subunits formed dimers with normal subunits in obligate heterozygotes (MPS VI+/b). In contrast, mutant isozyme a subunits from obligate MPS VIa/+ heterozygotes did not dimerize with the normal subunit, and the mutant and normal isozymes could be separated by anion exchange chromatography and polyacrylamide gel electrophoresis. Characterization of the partially purified residual hepatic arylsulfatase B activity from the heteroallelic homozygotes revealed the presence of both mutant isozymes a and b. The demonstration of two allelic mutations in the feline arylsulfatase B gene documented the occurrence of genetic heterogeneity in feline mucopolysaccharidosis VI and permitted characterization of the enzymatic defect in homoallelic and heteroallelic (genetic compound) homozygotes, providing a model for the study of allelism in human genetic disorders.