Evidence is emerging with regard to the role of tissue non-specific alkaline phosphatase (TNAP) in neural functions. As an ectophosphatase, this enzyme might influence neural activity and synaptic transmission in diverse ways. The localization of the enzyme in known neural circuits, such as the retina, might significantly advance an understanding of its role in normal and pathological functioning. However, the presence of TNAP in the retina is scarcely investigated. Our multispecies comparative study (zebrafish, cichlid, frog, chicken, mouse, rat, golden hamster, guinea pig, rabbit, sheep, cat, dog, ferret, squirrel monkey, human) using enzyme histochemistry and Western blots has shown the presence of TNAP activity in the retina of several mammalian species, including humans. Although the TNAP activity pattern varies across species, we have observed the following trends: (1) in all investigated species (except golden hamster), retinal vessels display TNAP activity; (2) TNAP activity consistently occurs in the photoreceptor layer; (3) in majority of the investigated species, marked TNAP activity is present in the outer and inner plexiform layers. In zebrafish, frog, chicken, guinea pig, and rat, TNAP histochemistry has revealed several sublayers of the inner plexiform layer. Frog, golden hamster, guinea pig, mouse, and human retinas possess a subpopulation of amacrine cells positively staining for TNAP activity. The expression of TNAP in critical sites of retinal signal transmission across a wide range of species suggests its fundamental, evolutionally conserved role in vision.