Previous studies have shown that embryonal carcinoma (EC) cells express the fibroblast growth factor k-FGF; however, there is a large decrease in the expression of this gene when EC cells differentiate. In addition, it has been shown that differentiation of mouse F9 EC cells reduces the expression of a reporter gene under the control of both the putative human k-FGF promoter and an enhancer-like element that is located in the third exon of the k-FGF gene. Given the low degree of sequence similarity between the human k-FGF gene and the murine k-FGF gene upstream of the transcription start site, it was unclear whether human sequences mimic fully the regulation of the k-FGF gene in mouse cells. To address this question, we have examined the expression of gene constructs containing various regions of the murine k-FGF gene in two mouse EC cell lines and one mouse embryonic stem (ES) cell line. Our results demonstrate that the mouse 5' flanking region, like the human 5' flanking region, cannot support expression of the reporter gene. In both EC cell lines and the ES cell line, expression of the reporter gene is elevated 10- to 100-fold by the addition of a 316-bp region taken from the third exon of the murine k-FGF gene. In addition, we provide evidence that octamer binding proteins are involved in the regulation of the k-FGF gene. Last, this study has identified regions upstream of the transcription start site that appear to regulate the expression of the murine k-FGF gene in EC cells and in ES cells.