Recent experiments by Fesenko et al and ourselves have shown that excised membrane patches from retinal rod outer segments contain a cyclic GMP-sensitive conductance which has electrical properties similar to those of the light-sensitive conductance. This finding supports the notion that cGMP mediates phototransduction (see ref. 3) by directly modulating the light-sensitive conductance. However, some uncertainty remained about whether the patch experiments had discriminated completely between plasma and intracellular disk membranes; thus the cGMP response in an excised membrane could have resulted from contaminating disk membrane fragments, which are known to contain a cGMP-regulated conductance. Furthermore, the patch conductance has not yet been shown to be light-suppressible, an ultimate criterion for identity with the light-sensitive conductance. We now report experiments on a truncated rod outer segment preparation which resolved these issues. The results demonstrated that the cGMP-sensitive conductance was present in the plasma membrane of the outer segment, and that in the presence of GTP the conductance could be suppressed by a light flash. With added ATP, the effectiveness of the light flash was reduced and the suppression was more transient. The effects of both GTP and ATP were consistent with the known biochemistry. From the maximum current inducible by cGMP, we estimate that approximately 1% of the light-sensitive conductance is normally open in the dark; this would give an effective free cGMP concentration of a few micromolar in the intact outer segment in the dark.