1. Patch-clamp recordings of single cyclic GMP-activated channels from toad rod photoreceptors were made in inside-out membrane patches containing only one such channel. Patches were obtained from the inner segment, where the density of cyclic GMP-activated channels is lower than in the outer segment, making one-channel patches possible. The dependence of channel gating on cyclic GMP concentration ([cyclic GMP]) was studied. At low [cyclic GMP] (5-10 microM), channel openings were infrequent and occurred as bursts of rapid opening and closing. As [cyclic GMP] was increased, bursts became more frequent, until at 1 mM the activity fused into long bouts of rapid flicker between open and closed states. 2. The duration of brief openings and closings (flicker) within bursts was not affected by [cyclic GMP]. This suggests that the rapid flicker within bursts results from an intrinsic channel property not associated with agonist-induced receptor activation. 3. At 10 microM-cyclic GMP, the distribution of closed times was fitted by a sum of three exponential components. The briefest, with time constant averaging 0.29 ms, corresponded to the brief closings within bursts, while the two longer components, with time constants averaging 3.5 and 32 ms, corresponded to much longer closings between bursts. At 0.5 or 1 mM-cyclic GMP (saturation), the longer components disappeared, and the distribution of closed times was fitted by a single-exponential equation with the same time constant as the briefest component observed at lower concentrations. 4. Because the channel continued to flicker even at high [cyclic GMP], the maximal probability of being in the open state (Po) did not approach 1.0, averaging 0.30 +/- 0.05 (N = 8). The relation between Po and [cyclic GMP] was fitted by the Hill equation with an exponent of 3, suggesting that binding of cyclic GMP to multiple sites is required to open the channel.