Xvent homeobox proteins are induced by BMP-4 signaling and have been known to mediate many BMP-4 activities as key downstream transcriptional factors. In order to investigate the regulatory mode of Xvent transcription, we isolated genomic DNA of the Xbr-1a/Xvent-2 containing the promoter region responsive to BMP-4 signaling. The cis-acting elements located within the Xbr-1a/Xvent-2 promoter and the regulation modes by BMP-4 signaling were analyzed by serial deletion and site-directed mutagenesis experiments. The upstream -235bp of the promoter retained the full transcriptional activity and BMP-4-response when compared with the longest promoter construct. Further analysis indicated that two separated 15 bp regions contained a strong positive element and BMP-4-response element. Site-directed mutagenesis of those regions suggests that those two regions cooperate for the promoter activity and BMP-4-response. Moreover, we found that the transcription factors, Oaz and PEBP2alphaA, were able to elicit additive effects with BMP-4 signaling on Xbr-1a/Xvent-2 reporter activities. These results indicate that transcriptional regulation of the Xbr-1a/Xvent-2 gene occurs in a complex mode through the cooperation of various transcription factors. (C) 2002 Elsevier Science (USA). All rights reserved.