The expression pattern and property profile of the neuronal Ca2+ sensor guanylate cyclase-activating protein 3 (zGCAP3) was studied by immunochemical approaches, biophysical methods and enzymatic assays. Using affinity purified antibodies immunoreactivity towards zGCAP3 was weakly detected in the outer and strongly in the inner segments of cone cells as well as in the outer plexiform layer, to a lesser degree also in the inner plexiform and ganglion cell layer of the zebrafish retina. This cellular distribution was independent of a dark/light cycle. Some neuronal Ca2+-sensors are acylated (mainly myristoylated) at the amino-terminus. Probing larval and adult stages of the developing zebrafish retina indicated that zGCAP3 was first expressed in a nonmyristoylated form, but was finally present in the adult retina as a myristoylated protein. While zGCAP3 did not undergo a classical Ca2+-myristoyl switch as investigated by surface plasmon resonance spectroscopy, myristoylation had two main other consequences: it enhanced the Ca2+-sensitivity of the Ca2+-induced conformational change and it stabilized the protein conformation. Differences between myristoylated and nonmyristoylated zGCAP3 were also observed in modulating the kinetic and catalytic parameters of the GCAP-target, a membrane bound guanylate cyclase. Thus, the stabilizing effect of the myristoyl group is apparently less important in the larval than in the adult fish.