ATP released by neurons and glia acts as a neurotransmitter and neuromodulator via specific purinergic receptors in the nervous system. Extracellular ATP concentrations are precisely regulated by ecto-nucleoside-triphosphate diphosphohydrolases (ENTPDases, ectonucleotidases). ATP hydrolysis products can also either act on specific purinergic receptors or be further metabolized extracellularly to adenosine, a potent inhibitory neuromodulator. We showed that two major subtypes of ectonucleotidases (ENTPDase type 1 and 2) are expressed in zebrafish and mouse retina both by western blot and immunohistochemistry. Both enzymes are heterogeneously distributed among retinal layers and principally expressed in the inner half of the retina. However, their expression pattern overlaps only partially because ENTPDase type 2 also exhibits a strong immunoreactivity on the outer plexiform layer. ENTPDases-like activity was also demonstrated on retinal membranes, by using radioactive nucleotides as substrates, in the presence of inhibitors of all other likely present enzymes with either ATPase or phosphatase activity. At present we are exploring ENTPDase subtypes expression on different cell types and synaptic layers throughout mouse and zebrafish retinas. To this end, we are comparing co-localization of immunoreactivity for both ENTPDase subtypes and several retinal cells and synaptic layers markers. In conclusion, the vertebrate retina contains at least two different types of ENTPDases with ATPase activity. These enzymes show a specific distribution on cell subtypes and synaptic retinal layers. Since ENTPDase 1 produces AMP in a much higher rate than ENTPDase 2, our results suggest a localized regulation of extracellular adenosine levels as well as ATP/ADP extracellular signaling.