Tryptophan hydroxylase is the first of four enzymes in the melatonin biosynthetic pathway. Recent studies have shown that there are two genes, Tph1 and Tph2, that encode tryptophan hydroxylase in mammals. In this study, we investigated which of the two genes is expressed in the rat retina. To that end, we measured Tph1 (classical Tph) and Tph2 mRNA levels using real-time quantitative RT-PCR in the retina. Our data demonstrate that Tph1 mRNA is the prevalent form expressed in the retina; Tph2 mRNA is also present but the level is very low. We also measured Tph1 expression levels in the outer nuclear layer, inner nuclear layer, and ganglion cell layer by combining laser capture microdissection and real-time RT-PCR. Tph1 mRNA is more abundant in the photoreceptors of the outer nuclear layer than in the inner nuclear layer or ganglion cell layer. Tph1 and Tph2 transcripts showed robust diurnal rhythms of abundance, with highest levels at night. Our results support the hypothesis that Tph1 is involved in melatonin synthesis in retinal photoreceptor cells.