AbstractDevelopment of the eye is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process may have been highly conserved during evolution. Herein we analyzed the promoter of the human βB1‐crytallin gene in zebrafish by transgenic experiments. To delineate the evolutionarily conserved regulatory elements, we performed serial deletion assays in the promoter region. The results demonstrated that the −90/+61‐bp upstream proximal promoter region is sufficient to confer lens‐tissue specificity to the human βB1‐crystallin gene in transgenic zebrafish. Through phylogenetic sequence comparisons and an electrophoretic mobility shift assay (EMSA), a highly conserved cis‐element of a six–base pair sequence TG(A/C)TGA, the consensus sequence for the Maf protein binding site, within the proximal promoter region was revealed. Further, a site‐mutational assay showed that this element is crucial for promoter activity. These data suggest that the fundamental transcriptional regulatory mechanism of the βB1‐crystallin gene has been well conserved between humans and zebrafish, and plausibly among all vertebrates, during evolution. Developmental Dynamics 235:435–443, 2006. © 2005 Wiley‐Liss, Inc.