The UV-sensitive cones of the zebrafish are insensitive to visible light and can therefore be used for Ca² measurements with fluo dyes and an argon ion laser without undue bleaching of the cone photopigment. This has made it possible for the first time to make multiple Ca² measurements from the same vertebrate photoreceptor (Leung et al., 2002). We have used this technique to explore in detail the relationship between outer segment Ca² concentration (Ca² _i) and photocurrent during a flash of light and in the presence of backgrounds. Dark-adapted zebrafish were stunned by concussion, then killed by decapitation and pithing. Cones were isolated from the retina and loaded for 30 min wtih 10 µM fluo 4-AM; fluorescence was measured as described previously (Matthews & Fain 2001, 2002). Simultaneous photocurrent measurements were made with a suction pipette.A single cone was stimulated repeatedly with a flash of constant intensity from the optical bench; after each light flash a single 50 ms laser exposure was used to evoke dye fluorescence before and at four times during the ensuing flash response. Response waveforms to the light flash alone before and after the fluorescence measurements were nearly identical, indicating that this protocol bleached a minimal amount of photopigment. Comparison of photocurrent with fluorescence indicated that Ca² declined after the flash and then recovered with a time course that was indistinguishable from that of the photocurrent. This indicates that Ca² _i in the cone outer segment is primarily determined by Ca² flux through the light-dependent channels (Sampath et al., 1999).We also exposed cones to steady background light and compared the change in sensitivity and photocurrent with the change in Ca² _i.For dim backgrounds, photocurrent and Ca² declined in proportion, but for brighter backgrounds, Ca² was considerably greater than expected, probably as the result of light-induced Ca² release (Leung et al., 2002; Brockerhoff et al., 2003). This release of Ca² seemed, however, to have little effect on the sensitivity of the cell which declined monotonically according to the Weber-Fechner relation in spite of the anomalous increase in Ca² _i,suggesting that changes in Ca² _i may have only a small effect on light adaptation in cones apart from the modulation of guanylyl cyclase.