Numerous reports have concluded that zebrafish (Danio rerio) possesses A1-based visual pigments in their rod and cone photoreceptors. In the present study, we investigated the possibility that zebrafish have a paired visual pigment system. We measured the spectral absorption characteristics of photoreceptors from zebrafish maintained in different temperature regimes and those treated with exogenous thyroid hormone using CCD-based microspectrophotometry. Rods from fish housed at 15°C and 28°C were not significantly different, having λmax values of 503 ± 5 nm (n = 106) and 504 ± 6 nm (n = 88), respectively. Thyroid hormone treatment (held at 28°C), however, significantly shifted the λmax of rods from 503 ± 5 nm (n = 194) to 527 ± 8 nm (n = 212). Cone photoreceptors in fish housed at 28°C (without thyroid hormone treatment) had λmax values of 361 ± 3 nm (n = 2) for ultraviolet-, 411 ± 5 nm (n = 18) for short-, 482 ± 6 nm (n = 9) for medium-, and 565 ± 10 nm (n = 14) for long-wavelength sensitive cones. Thyroid hormone treatment of fish held at 28°C significantly shifted the λmax of long-wavelength sensitive cones to 613 ± 11 nm (n = 20), substantially beyond that of the λmax of the longest possible A1-based visual pigment (∼580 nm). Thyroid hormone treatment produced smaller shifts of λmax in other cone types and increased the half-band width. All shifts in photoreceptor λmax values resulting from thyroid hormone treatment matched predictions for an A1- to A2-based visual pigment system. We therefore conclude that zebrafish possess a rhodopsin–porphyropsin interchange system that functions to spectrally tune rod and cone photoreceptors. We believe that these observations should be carefully considered during analysis of zebrafish spectral sensitivity.