Lysosomal membranes contain two highly glycosylated proteins, designated as lamp-1 and lamp-2, as major components. Lamp-1 and lamp-2 are similar to each other in the protein structure. Here, we investigated the biosynthetic transport of lamp-2 through the endocytic vacuoles in cultured rat hepatocytes in comparison with that of lamp-1, which has previously been studied [Akasaki et al. (1995) Exp. Cell Res. 220, 464-473]. Newly synthesized lamp-2 (NS-lamp-2) was transported to the trans-Golgi from rough endoplasmic reticulum with a half time (t1/2) of 32 min, more slowly than NS-lamp-1 (t1/2 = 13 min). After leaving the trans-Golgi, NS-lamp-2 is transferred to at least three compartments; the cell surface (t1/2 = 47 min), cell peripheral early endosomes (t1/2 = 38 min) and perinuclear late endosomes (t1/2 = 48 min). NS-lamp-2 transported to any compartment is delivered finally to lysosomes (t1/2 = 90 min). A significant fraction of NS-lamp-2 (45% of the total) was transported from the trans-Golgi to early endosomes, and then delivered to dense lysosomes via perinuclear late endosomes, whereas a major portion of NS-lamp-1 follows an intracellular route to late endosomes without passing through the cell periphery. NS-lamp-2 leaves the cell peripheral region more rapidly than NS-lamp-1. The kinetic and quantitative data for biosynthetic transport of NS-lamp-2 to early endosomes and the cell surface indicate that NS-lamp-2 may be transported first to early endosomes, from which a small portion of it (approximately 3.5% of the total) moves to the plasma membrane via a recycling system. In contrast, a small fraction of NS-lamp-1 is transported to the plasma membrane directly from the trans-Golgi, since NS-lamp-1 is delivered to the plasma membrane and early endosomes with almost the same half times.