Light adaptation in rod photoreceptors is thought to involve down-regulation of the signaling activity of photoactivated rhodopsin (R*). However, electrophysiological evidence in support of this notion has come largely from studies of truncated, perfused rod outer segments and of rods genetically engineered to perturb known steps in R* deactivation. To test this hypothesis within intact native rods, we examined the effect of a fixed conditioning flash on rods prepared to contain 9-demethyl rhodopsin (9dR) in addition to residual rhodopsin. 9dR, an opsin-based photopigment containing 11-cis 9-demethylretinal as its chromophore, exhibits a blue-shifted excitation spectrum and sluggish deactivation kinetics, properties that distinguish the signaling activities of photoactivated 9dR (9dR*) from those of R*. Saturating photocurrent responses mediated preferentially by R* and 9dR* were obtained with test flash stimulation at 640 and 440 nm, respectively, under dark-adapted conditions (unconditioned response) and at a fixed time after a 640-nm conditioning flash of fixed high intensity. At each test wavelength, the decrease in photocurrent saturation period induced by the conditioning flash was analyzed to determine ψ, the sensitivity of the conditioned response relative that of the unconditioned response; ψ640 /ψ440, the ratio of relative sensitivities, was then obtained. Data obtained from 12 rods yielded ψ640 /ψ440 = 0.60 ± 0.13 (mean ± SD). As common pools of transducin and other downstream components mediate transduction initiated by both R* and 9dR*, the finding that ψ640 < ψ440 provides direct evidence for the down-regulation specifically of R*'s signaling activity during rod light adaptation.