The rat βB2‐crystallin gene is active only during a specific stage of the differentiation of rat lens fibre cells directed by basic fibroblast growth factor. The regulatory elements that determine the transient activity of this gene are located in the ‐750/‐123 region and in the first intron. Singly, these elements act as silencers, together they constitute an enhancer that is active only during the specific differentiation stage. An additional silencer is found between ‐123 and ‐77. The proximal promoter region contains a Pax‐6 binding site at ‐65/‐51. In vitro, binding to this site could be detected but, according to in vivo footprinting experiments, this site is not occupied in the endogenous gene. Furthermore, co‐expression of Pax‐6 did not enhance promoter activity. Finally, mutation or deletion of this site did not affect promoter activity: the region ‐37/+10 sufficed for basal promoter activity. The cooperation between the ‐750/‐123 region and the first intron of the βB2‐crystallin gene not only determines the differentiation stage‐specific activity of the gene, but also contributes to the highly increased expression in lens cells compared with non‐lens cells.