Kinesin is a microtubule-based motor protein involved in organelle transport in neuronal and nonneuronal cells. Although a single kinesin motor has been thought to serve all cell types, we document here that neurons express a second conventional kinesin heavy chain (nKHC) that is 65% identical in amino acid sequence to the ubiquitously expressed kinesin heavy chain (uKHC). By preparing antibodies which distinguish between the two KHCs, we demonstrate that nKHC is a nucleotide-dependent microtubule-binding protein which partially cofractionates with membrane organelles. Immunolocalization experiments show that nKHC is distributed throughout the CNS but is highly enriched in subsets of neurons. In hippocampal neurons in culture, uKHC is distributed uniformly throughout the neuron, whereas nKHC is selectively concentrated in the cell body. These results demonstrate that mammalian neuronal tissue contains two conventional kinesin motors which may serve distinct functions in microtubule-based transport.