Expression of the laminin B1 gene is known to be induced late during the differentiation of F9 cells by retinoic acid (RA) and dibutyryl cAMP. The involvement of retinoic acid receptors (RARs) has been demonstrated recently in the late induction of laminin B1 gene expression, although the precise regulatory mechanism is not known. In this study, we have reconstituted an efficient in vitro transcription system using F9 nuclear extracts and defined the core promoter structure of the murine laminin B1 gene. The laminin B1 gene was shown to lack a TATA box. The level of the in vitro transcription of the laminin B1 gene was determined by at least three regions between the transcription initiation sites and -100. The most distal region (from -89 to -69) contained three GC boxes. The second region (from -62 to 47) contained a direct repeat of TG(C/A)GCA motif. The proximal region (from -45 to -11) contained another direct repeat of CCTCCCT(C/A)GG motif. A deletion of any one of the three regions respectively decreased the level of transcription to about 20% of wild type DNA. The protein binding analyses revealed that F9 cells contain a factor(s) binding to the TG(C/A)GCA repeat, which was also found in HeLa cells. Together with the observation that the 5' ends of the laminin B1 mRNA from the differentiated F9 cells were identical to those from the undifferentiated F9 cells, it was concluded that the three regions identified here constitute the core promoter of the laminin B1 gene.