Rab GTPases play an important regulatory role in early endocytosis. We recently demonstrated that epitope-tagged Rab15 (HArab15) co-localizes with Rab4, -5, and -11 on early endosomal membranes in CHO cells (Zuk, P. A., and Elferink, L. A. (1999) J. Biol. Chem. 274, 22303-22312). To characterize the role of Rab15 in endocytosis, we prepared functional mutants of HArab15 and examined their effects on early endocytic trafficking. Wild-type HArab15 and its constitutively active, GTP-bound mutant (Q67L) reduce fluid phase and receptor-mediated endocytosis without affecting the rate of recycling from early endosomal compartments. Inhibition of early endocytosis appears to be due to a reduction in the rate of homotypic early endosome fusion. Conversely, mutations that constitutively inactivate HArab15 stimulate early endocytosis and the homotypic fusion of early endosomes in vitro. Unlike active forms of HArab15, constitutively inactive HArab15 mutants also affect recycling from early endosomal compartments. Moreover, the two constitutively inactive mutants, GDP-bound HArab15-T22N and the non-nucleotide binding mutant HArab15-N121I, differentially regulate the transit of fluid phase and receptor-mediated endocytic tracers through early/sorting endosomes. Together, these data suggest that HArab15 may counteract the reported stimulatory effect of Rab5 on early endocytosis. Consistent with this, overexpression of constitutively active HArab15-Q67L attenuates Rab5-stimulated endocytosis, whereas Rab5-stimulated endocytosis is augmented in cells overexpressing a constitutively inactive HArab15 mutant defective in guanine nucleotide binding (N121I). Our data indicate that HArab15 differentially regulates distinct steps in membrane trafficking through early/sorting and pericentriolar recycling endosomes.