Tyrosinase-related protein-1 (TRP-1) is a 75 kDa type-1 transmembrane glycoprotein localized to the melanosome. The mechanism by which newly synthesized TRP-1 reaches its ultimate destination is currently unknown, but has been speculated to occur via the endosomal pathway. Recently, it has been shown that phosphatidylinositide (PI) 3-kinase is involved in various cellular functions, including regulating the constitutive movement of proteins from one intracellular compartment to another; however, whether PI 3-kinase participates in the trafficking of proteins such as TRP-1 to the melanosome is unknown. In this study we investigate the role of PI 3-kinase on the trafficking of TRP-1 in human melanoma MeWo cells using wortmannin, a potent inhibitor of PI 3-kinase. Our investigations demonstrate that wortmannin interferes with the membrane trafficking of TRP-1 in MeWo cells, and that it specifically results in the redistribution of the protein within a novel vesicular compartment with characteristics of the endosomal and lysosomal compartments [positive for LAMP-1, and partially positive for CD63 and cation-independent mannose 6-phosphate receptors (CI-M6PR)], and is accessible to internalized proteins such as immunoglobulins. Movement within this novel compartment is microtubule and GTPase dependent. These findings have led us to postulate that TRP-1 is sorted from the trans-Golgi network to a compartment in the vicinity of late endosomes, trafficking from which to the melanosome appears to be dependent on PI 3-kinase as it is blocked by wortmannin.