We have characterized a gene for mouse S-antigen and compared its sequence with that of corresponding human and two recently published Drosophila S-antigen genes. The mouse S-antigen gene was approximately 50 kbp in length and consisted of 16 exons and 15 introns. The length of most exons was less than 100 bp and the smallest one was only 10 bp. In contrast, the length of most introns was larger than 2 kbp and the gene consisted of 97% intron and 3% exon. Both splice sites for donor and accepter were in good agreement with the GT/AG rule. S-antigen genes in human and mouse were highly conserved. In contrast, genes for the Drosophila 49-kDa arrestin homolog and arrestin consist of three introns and four exons and two introns and three exons, respectively. The 5'-flanking region of the mouse S-antigen gene, approximately 1.0 kbp long, had no regulatory elements for transcription such as the TATA, CAAT and GC boxes, while a Drosophifu arrestin gene has TATA and CAAT boxes. Interestingly, the 5'-flanking region of the mouse gene had promoter activity in an in vitro transcription assay using a nuclear extract of rat brain. A major transcription start site was found at 387 bp upstream from the translation start codon ATG in mouse. From our results, and those of others, we suggest that the S-antigen gene has evolved from a coinman ancestor gene by either insertion or deletion of introns. Such an alteration of gene structure may have played a role in the evolution of the S-antigen.Phototransduction, which converts light energy into neuronal impulse, takes place in the photoreceptor cells of the retina. Initially, light activates rhodopsin, the photoactivated rhodopsin then interacts with transducin, which in turn activates cGMP phosphodiesterase which then hydrolyzes cGMP. The decrease in intracellular concentration of cGMP modulates the influx of Na' ions through plasma-membrane channels and initiates membrane hyperpolarization [I, 21. Shortly after light activation, rhodopsin is phosphorylated by rhodopsin kinase and this modification is thought to be involved in deactivation of phototransduction [3, 41. In contrast to the mammalian system, Drosophila phototransduction differs from that of vertebrates. Phospholipase C, instead of phosphodiesterase, catalyzes the hydrolysis of phosphatidyl inositol 4,5-bisphosphate to inositol trisphosphate [5].One of the major soluble proteins in photoreceptor cells, S-antigen, has been well characterized [6]. This protein is also referred to as 48-kDa protein or arrestin [4,7, 81 and is known to have an inhibitory role in the activated phototransduction cascade [3,8]. Although the exact mechanism of the inhibition is unknown, it is postulated that S-antigen binds to photoexcited, phosphorylated rhodopsin and quenches the activation of light-dependent cGMP phosphodiesterase [3, 7, 9, Corre.~pondencc to