ATP7B is a copper-transporting P-type ATPase present predominantly in liver. In basal copper, hepatic ATP7B is in a post-TGN compartment where it loads cytoplasmic Cu(I) onto newly-synthesized ceruloplasmin. When copper levels rise, the protein redistributes via unique vesicles to the apical periphery, where it exports intracellular Cu(I) into bile. We want to understand the mechanisms regulating ATP7B's copper-sensitive trafficking. Earlier, we reported the presence of apical targeting/TGN retention signal(s) within residues 1-63 of human ATP7B; deletion of these residues resulted in a mutant protein that was not efficiently retained in the post-TGN in low copper and constitutively trafficked to the basolateral membrane of polarized, hepatic WIF-B cells +/- copper (13). In this study, we used mutagenesis and adenovirus infection of WIF-B cells followed by confocal immunofluorescence microscopy analysis to dissect the signal in the context of full-length ATP7B. We also analysed the expression of selected mutants in livers of copper-deficient and -loaded mice. Our combined results clearly demonstrate that nine amino acids, F₃₇AFDNVGYE45, comprise an essential apical targeting determinant for ATP7B in elevated copper and participate in the protein's TGN retention under low copper conditions. The signal is novel, does not require phosphorylation and is highly conserved in ~24 species of ATP7B. Furthermore, N41S, which is part of the signal we identified, is the first and only Wilson Disease-causing missense mutation in residues 1-63 of ATP7B. Expression of N41S-ATP7B in WIF-B cells severely disabled the protein's targeting and retention. We present a working model of how this physiologically-relevant signal might work.