cDNA clones encoding the β-subunit of the photoreceptor cGMP phosphodiesterase (PDE) were isolated from a human retina library and their sequence was determined. The encoded polypeptide consists of 854 amino acid residues with a calculated molecular mass of 98,416 Da. Alignment of the deduced amino acid sequence with the earlier analysed α-, β- and α'-subunits of bovine and mouse PDEs demonstrates a high homology. Two overlapping recombinant λ phage clones containing 26 kb of the human PDE β-subunit gene were isolated from the genomic library. A total nucleotide sequence of exons 4–22 of the PDE β-subunit gene was established which completely corresponded to the cDNA structure. According to sequence analysis no potential possibility for alternative splicing of the β-subunit gene was observed between exons 20 and 21 which led to the formation of the β'-subunit as described for mouse PDE. Polymerase chain reaction (PCR) experiments also confirm the absence of the PDE β'-subunit in human retina.