We have cloned full-length human and mouse cDNAs of ABCB9, which encodes a predicted multiple-spanning transmembrane domain and a nucleotide-binding domain with Walker motifs. It is therefore designated as a "half" ATP binding cassette (ABC) transporter. Northern analysis shows that the ABCB9 mRNA is expressed at a high level in testes and moderate levels in brain and spinal cord. A splice variant mRNA deleted in the last pair of predicted transmembrane segments was shown to be expressed in human tissues. Phylogenetic analysis indicates that ABCB9 is closely related to TAP1 and TAP2, two "half" ABC proteins found in endoplasmic reticulum. ABCB9 protein colocalized with the lysosomal markers, LAMP1 and LAMP2, in transfected cells. ABCB9 protein appears to be most highly expressed in the Sertoli cells of the seminiferous tubules in mouse and rat testes. These cells have high levels of phagocytosis and secretory activities. These findings pave the way for further investigation into the potential novel function of ABCB9 in lysosomes.