Rpe65 knockout mice (Rpe65-/-) are unable to synthesize the visual pigment chromophore 11-cis retinal; however, if these animals are reared in complete darkness, the rod photoreceptors accumulate a small amount of 9-cis retinal and its corresponding visual pigment isorhodopsin. Suction-electrode recording of single rods from dark-reared Rpe65-/- mice showed that the rods were about 400 times less sensitive than wild-type control rods and that the maximum responses were much smaller in amplitude. Spectral sensitivity measurements indicated that Rpe65-/- rod responses were generated by isorhodopsin rather than rhodopsin. Sensitivity and pigment concentration were compared in the same mice by measuring light responses from rods of one eye and pigment concentration from the retina of the other eye. Retinas had 11-35% of the normal pigment level, but the rods were of the order of 20-30 times less sensitive than could be accounted for by the loss in quantum catch. This extra desensitization must be caused by opsin-dependent activation of the visual cascade, which leads to a state equivalent to light adaptation in the dark-adapted rod. By comparing the sensitivity of dark-reared Rpe65-/- rods to that produced in normal rods by background light, we estimate that Rpe65-/- opsin is of the order of 2.5x10(-5) as efficient in activating transduction as photoactivated rhodopsin (Rh*) in WT mice. Dark-reared Rpe65-/- rods are less desensitized than rods from cyclic light-reared Rpe65-/- mice, have about 50% more photocurrent and degenerate at a slower rate. Retinas sectioned after 9 months in darkness show a larger number of photoreceptor nuclei in dark-reared animals than in cyclic light-reared animals, though both have fewer nuclei than in cyclic light-reared wild-type retinas. Both also have shorter outer segments and a lower free-Ca2+ concentration. These experiments provide the first quantitative measurement of opsin activation in physiologically responding mammalian rods.