1. Gene expression profiles in neural stem cell differentiation in vitro were determined by cDNA microarray analysis. 2. Total RNA was extracted and reverse transcripted into cDNA from differentiated and undifferentiated neural stem cells. The 33P labeled cDNA was hybridized with a cDNA microarray consisting of 14,000 human genes. 3. The results showed that a total of 1406 genes were differentially expressed, of which 148 genes exhibited more than twofold differences. Some genes were obviously activated while others were strongly repressed. These changes in gene expression suggest that differentiation is regulated by different genes at different expressional levels. By biological classification, the differentially expressed genes were divided into four functional categories: molecular function, biological process, cellular component, and new functional genes or ESTs. 4. These findings will be a valuable contribution for gene expression profiling and elucidation of neural stem cell differentiation mechanisms.