AP-3 is a heterotetrameric protein complex involved in intracellular vesicle transport. Molecular analyses show that Ap3b1, which encodes the AP-3 β3A subunit, is altered in pearl mice. To provide genetic evidence that mutation of Ap3b1 is responsible for the pearl phenotype and to determine the null phenotype, the Ap3b1 gene was disrupted by homologous recombination. Mice homozygous for the resulting allele, Ap3b1^LN, or compound heterozygotes with pearl, displayed phenotypes similar to those of pearl mice, confirming that Ap3b1 is the causal gene for pearl. Moreover, pearl is likely to be a hypomorph as the Ap3b1^LN homozygotes had a lighter coat color and accumulated fewer of the μ3 and δ3 subunits of AP-3 than did pearl mice. Finally, immunofluorescence analysis of fibroblasts and melanocytes cultured from Ap3b1^LN homozygotes revealed that the lysosomal membrane proteins Lamp I and Lamp II and the melanosomal membrane protein tyrosinase were mislocalized. In particular, the Lamp proteins were clustered on the cell surface. These findings strengthen the evidence for an alternate pathway via the plasma membrane for cargo normally transported to organelles by AP-3.