A heterotetrameric complex termed AP-3 is involved in signal-mediated protein sorting to endosomal-lysosomal organelles. AP-3 has been proposed to be a component of a nonclathrin coat. In vitro binding assays showed that mammalian AP-3 did associate with clathrin by interaction of the appendage domain of its beta 3 subunit with the amino-terminal domain of the clathrin heavy chain. The beta 3 appendage domain contained a conserved consensus motif for clathrin binding. AP-3 colocalized with clathrin in cells as observed by immunofluorescence and immunoelectron microscopy. Thus, AP-3 function in protein sorting may depend on clathrin.