Ion channels in excised patches of plasma membrane are generally considered to be isolated from any intracellular regulation mechanisms. For example, in excised patches of vertebrate rod outer segment plasma membrane, the cGMP-activated cation channels have traditionally been studied in room light because the enzyme cascade linking photon absorption to channel closure was assumed to be inoperative. To investigate the possibility that, in fact, such excised patches retain a functional phototransduction enzymatic cascade, this same preparation was studied in darkness. Patches excised in the dark were found to retain the light sensitivity of their cGMP-induced conductance and the ability to synthesize cGMP. In the presence of guanosine 5'-[y-thioltriphosphate (GTP[yS]), a nonhydrolyzable GTP analog, light suppresses the cGMP-induced conductance irreversibly. Furthermore, inhibitors of phosphodiesterase activity reduce light sensitivity, whereas activated phosphodiesterase or activated transducin does not directly affect the channels. These results (i) establish that excised patches from rod outer segment retain functional phototransduction enzymes, (it) support the classical view that channel opening is modulated by phosphodiesterase-mediated cGMP hydrolysis, and, most surprisingly, (i) demonstrate that diffusion in excised patches is so restricted that local enzymes can induce variations in the concentration of small molecules. The indication that excised patches are not as simple as usually surmised opens the possibility of using them to study other intracellular transduction mechanisms.In the traditional representation of excised patches, membrane proteins are considered to be isolated from any intracellular regulation mechanism involving either soluble enzymes or diffusible messengers. This isolation is presumed to arise from two phenomena: (i) soluble enzymes wash out from the patch when it is excised from the rest of the cell, and (ii) any intracellular messengers that might be synthesized by membrane enzymes diffuse instantaneously out into the "infinite" volume of the perfusion bath. In most cases, however, no attempt has been made to investigate the validity of these assumptions. The work reported here examines this problem in the specific case of excised patches of vertebrate rod outer segment (ROS) plasma membrane.Vertebrate vision is initiated in photoreceptor outer segments by a process that links the absorption of photons to the closure of ion channels in the plasma membrane (1,2 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.