The cytoplasmic tail of the H,K-ATPase β-subunit contains a putative tyrosine-based motif that directs the β-subunit's basolateral sorting when it is expressed in Madin-Darby Canine Kidney (MDCK) cells. When expressed in LLC-PK1 cells, however, the β-subunit is localized to the apical membrane. Several proteins that contain tyrosine-based motifs, including the low-density lipoprotein and transferrin receptors, show a similar sorting ‘defect’ when expressed in LLC-PK1 cells. For low-density lipoprotein and transferrin receptors, this behavior is due to the differential expression of the μ1B subunit of the AP-1B clathrin adaptor complex. μ1B is expressed by MDCK cells, but not LLC-PK1 cells, and transfection of μ1B into LLC-PK1 cells restores basolateral localization of low-density lipoprotein and transferrin receptors. For the β-subunit, however, μ1B expression in LLC-PK1 cells does not induce its basolateral expression. We found that the β-subunit interacts with both μ1B and μ1A in vitro and in vivo. The capacity to participate in a μ1B interaction therefore is not sufficient to program the β-subunit's basolateral localization in MDCK cells. Our data suggest that the H,K-ATPase β-subunit's basolateral sorting signal is either masked in certain epithelial cells, or requires an interaction with sorting machinery other than AP-1B for delivery to the basolateral plasma membrane.