Rhodopsin genes in most vertebrate species, with the exception of teleost fishes, contain introns. Despite differences in the gene structure, similar regulatory motifs have been identified in fish, amphibian and mammalian rhodopsin promoters, suggesting that rhodopsin gene regulation may be conserved in vertebrates. However, there is no direct evidence to support this notion. To address this, the rhodopsin promoter from the pufferfish, Fugu rubripes, was isolated and tested in transgenic mice and frogs. A 6.5 kb Fugu genomic fragment containing the rhodopsin gene and 4.5 kb 5' flanking region was able to direct expression of the Fugu rhodopsin gene to the retina of transgenic mice. In transgenic tadpoles, photoreceptor rod cell-specific expression of a reporter gene was achieved using only 500 bp Fugu rhodopsin promoter fragment. Mutagenesis of this promoter fragment revealed that a conserved NRE-like motif is crucial for the retina-specific expression. Our investigation suggests that the regulation of retinal specific expression is conserved in the pufferfish, frog and mouse and that the ancestral intron-containing rhodopsin gene has been displaced by an intronless copy in teleosts.