Accumulation of vitronectin protein increased in the conditioned medium of mouse neuroblastoma Neuro2a cells during retinoic acid-induced differentiation. To study the regulatory mechanism of the increase in vitronectin expression during the differentiation, the activity of the -527/+95 vitronectin promoter was observed in Neuro2a cells with or without retinoic acid treatment. The result showed that the -527/+95 promoter activity increased 2.7-fold with retinoic acid, and despite deletion of regions from -527 to -49 and +54 to +95 base pairs (bp), the -48/+53 promoter preserved the retinoic acid response. We recently showed that the -48/+53 region has two transcription factor Foxa (HNF3)-binding sites (site A from -34 to -25 bp and site B from +15 to +26 bp), suggesting that Foxa may up-regulate the vitronectin expression. Therefore, we examined the change of Foxa expression in Neuro2a cells during the differentiation. The expression of Foxa1 protein was increased during the differentiation, but the expression of Foxa2 protein was not detected. In addition, overexpression of Foxa1 increased the amount of vitronectin protein in the conditioned medium of Foxa1-overexpressed Neuro2a cells, but overexpression of Foxa2 only weakly increased it. The site-A and -B double mutation of the -527/+95 promoter remarkably reduced the promoter activity induced by Foxa overexpression, indicating that Foxa-binding sites in the -527/+95 region are located only on sites A and B. The mutation of site A in the -48/+53 promoter did not affect the retinoic acid response, but the site-B mutation abolished the constitutive promoter activity and remarkably reduced the promoter activity with retinoic acid. These results demonstrate that Foxa up-regulates the vitronectin expression during the retinoic acid-induced differentiation in Neuro2a cells.