Class V and VI myosins, two of the six known classes of actin-based motor genes expressed in vertebrate brain (Class I, II, V, VI, IX, and XV), have been suggested to be organelle motors. In this report, the neuronal expression and subcellular localization of chicken brain myosin V and myosin VI is examined. Both myosins are expressed in brain during embryogenesis. In cultured dorsal root ganglion (DRG) neurons, immunolocalization of myosin V and myosin VI revealed a similar distribution for these two myosins. Both are present within cell bodies, neurites and growth cones. Both of these myosins exhibit punctate labeling patterns that are found in the same subcellular region as microtubules in growth cone central domains. In peripheral growth cone domains, where individual puncta are more readily resolved, we observe a similar number of myosin V and myosin VI puncta. How-ever, less than 20% of myosin V and myosin VI puncta colocalize with each other in the peripheral domains. After live cell extraction, punctate staining of myosin V and myosin VI is reduced in peripheral domains. However, we do not detect such changes in the central domains, suggesting that these myosins are associated with cytoskeletal/ organelle structures. In peripheral growth cone domains myosin VI exhibits a higher extractability than myosin V. This difference between myosin V and VI was also found in a biochemical growth cone particle preparation from brain, suggesting that a significant portion of these two motors has a distinct subcellular distribution.