Melanophores offer an outstanding system for the study of intracellular motility. These cells aggregate their pigment-filled melanosomes to the cell center or disperse them throughout the cytoplasm in response to hormonal modulation of intracellular cyclic AMP levels in order to effect color changes in lower vertebrates [1]. Previous work from our laboratory demonstrated a role for microtubule-based motors in melanosome transport and we succeeded in reconstituting their regulated motility along microtubules in vitro [2,3]. Here we demonstrate that, in addition to microtubule-mediated motility, melanosomes purified from Xenopus melanophores exhibit unidirectional movement along actin filaments in vitro as well. Immunoblotting analysis shows that these organelles possess the actin-based organelle motor, myosin-V. In vivo, melanosomes are able to slowly disperse in the absence of microtubules, and this slow dispersion requires the integrity of the actin cytoskeleton. Furthermore, in cells with dispersed pigment, disruption of filamentous actin induces a rapid, microtubule-dependent aggregation of melanosomes to the cell center. Our results, together with the accompanying paper by Rodionov et al. [4], demonstrate that the concerted efforts of both microtubule-based and actin-based motors are required for proper melanosome distribution in melanophores. This is the first example of a biochemically defined organelle in possession of both plus-end and minus-end directed microtubule motors and a myosin; coordinated activity of all three motors is essential for organelle motility in vivo.