Liver 13-glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of 13-glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenie line B6 • PAC-Gus". Liver [3glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of 13-glucuronidase in Gusn/Gus" mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. [3-Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal fl-glucuronidase. The loss of liver lysosomal ~-glueuronidase activity was shown by immunotitration to be due to a decrease in the number of l3-glucuronidase molecules in lysosomes of the congenic strain. The Gus" structural alteration likely causes the lowered lysosomal 13-glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gtlsn/GUS b animals had intermediate levels of liver ~-glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus"/Gus" mice. Gusn is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.