The goal of this study was to extend the results of previous immunoassay, immunocytochemistry, and in situ hybridization studies showing the presence of insulin-related peptide in the rat retina by confirming the expression of insulin genes in the rat eye. Total and poly (A)+ RNA were isolated from whole rat eyes, and separately from the retina, choroid, iris, lens, and vitreous. The poly (A)+ RNA was used for preparation of insulin-specific cDNA according to a coupled reverse transcription polymerase chain reaction (RT-PCR) protocol under high stringency conditions. Southern transfers, restriction fragment analyses, and nucleotide sequencing were used to characterize and identify the amplified cDNA products. Amplified cDNA fragments of 329+/-6 base pairs (bp) were derived from whole rat eye and rat retina poly (A)+ RNA, but not from other regions of the eye. Southern blots probed with preproinsulin-specific primer demonstrated homology with similar-sized cDNA from rat pancreas. Restriction digests with 10 restriction enzymes and direct nucleotide sequencing confirmed that the 329-bp cDNA was identical to the previously known coding sequence for rat pancreatic preproinsulin1 DNA. The identification of retinal preproinsulin1 mRNA was confirmed. This correlates with previous studies showing insulin immunoreactivity in rat eyes and in cultured retina, and verifies in situ hybridization evidence for the presence of insulin-related mRNA in retinal glial cells.