Second messengers are involved in a number of cellular responses to a variety of stimuli. Diffusion of these second messengers likely will determine the speed and efficiency of such responses. Localization, particularly in large cells, would enhance the efficiency of such transduction systems by restricting the volume in which this diffusion takes place and thereby limiting the diffusion of soluble messengers. Phototransduction in Limulus ventral photoreceptors involves second-messenger systems; the volume of this cell is quite large, but the effect of a single photoexcited rhodopsin molecule is exerted over light-dependent channels localized within a very small area of the plasma membrane. In order to investigate localization of phototransduction in these photoreceptors, we have compared the light responses of small vesicles (photoballs) excised from these cells with those of the intact photoreceptors. We found that the basic kinetics of excitation and adaptation of the photoballs are essentially identical to those of the intact cell. This indicates that all of the necessary machinery for phototransduction is present and intact in the photoball and that any diffusion of second messengers that affect the normal light response of the cell must occur within a region at least as small as our photoballs (on the order of 1 micron3).