αA-Crystallin is a member of the small heat shock protein family that is abundantly expressed as a structural component in the vertebrate eye lens. In lenses of rodents and some other mammals, there occurs a minor variant of αA-crystallin, which has an insertion of 23 amino acid residues. This variant, αAins-crystallin, results from differential integration of an optional exon into a small fraction of the mRNA. We have studied whether this alternative splicing is caused by a non-consensus cytosine in the 5′ splice site adjacent to the optional exon. After replacement of the aberrant cytosine in the hamster αA-crystallin gene by a consensus thymine, and transient transfection of this gene in Chinese Hamster Ovary cells, the optional exon is indeed almost completely spliced into the mature mRNA. In contrast, replacement of the cytosine by adenine or guanine completely abolishes the splicing of the optional exon. Our results confirm that alternative splicing of the αA-crystallin primary transcript is mainly due to a non-consensus 5′ splice site nucleotide. However, we conclude that the small size of the optional exon is probably an additional contributing factor and therefore it seems that the splicing mechanism is based on recognition of exons rather than introns.