Ornithine-delta-aminotransferase (OAT) is a nuclear-encoded, mitochondrial enzyme that converts ornithine to glutamate semialdehyde. Although OAT is expressed in most tissues of the rat, liver, kidney, and retina have the highest levels of OAT activity. Studies of OAT regulation in liver and kidney have indicated transcriptional and translational controls for the enzyme in a tissue-specific manner. Little is known about OAT modulation in retinal tissue, although chorio-retinal degeneration is the predominant feature in a hereditary disorder of OAT deficiency, gyrate atrophy. To characterize OAT regulation in retinal lines, we studied its synthesis in two retinoblastoma strains, Y79 and RB355. Baseline OAT mRNA levels were similar in the two cell lines, yet Y79 expressed 3-fold more immunoreactive OAT protein and enzyme activity than RB355; this finding suggested the presence of a post-transcriptional mechanism for the regulation of steady-state OAT levels. Treatment of the two strains with estradiol or thyroid hormone for 24 h resulted in approximately 5-fold increases in OAT protein and activity. Since similar increases in OAT mRNA levels were observed in both strains after Northern blotting, it is likely that these hormones exert their effects at the transcriptional level. Finally, primer extension analysis revealed two OAT mRNA species in both strains, due to the presence of an additional exon (exon 2) in one of the transcripts. The absence of this exon in other tissues reflects the unique mechanisms which govern OAT in retinoblastomas.