The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and beta(beta)-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and beta-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, beta-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas beta-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the beta-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and beta-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and beta-III-tubulin during the neural differentiation of H9 cells. (C) 2013 Elsevier Inc. All rights reserved.