To assess the effects of pharmacologic agents on metabolite levels in identified retinal layers we have devised an apparatus that allows suprafusion of everted eye cups (vitreal surface exposed) or isolated retinas (photoreceptor surface exposed), recording of electroretinograms (ERG), and rapid freezing in a configuration which permits obtaining frozen tangential sections of the retina. Samples from identified layers of the freeze-dried sections can be subsequently weighed and processed for biochemistry by Lowry methods (Lowry and Passonneau , 1972), or cyclic nucleotides can be extracted from weighed samples and assayed. To test the usefulness of the apparatus, we have compared levels of guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP), measured in defined layers of suprafused rabbit retinas, to concentrations reported for retinal layers from eyes removed from light- and dark-adapted rabbits and quickly frozen. For most layers the concentrations of cyclic nucleotides were similar in both the suprafused and whole eye preparations. In experimental applications we suprafused dark-adapted eye cups and retinas with a physiological saline whose free calcium level was sharply reduced by chelation, and observed that cGMP levels were elevated only in those retinal layers containing photoreceptor portions. Ethyleneglycol bis-(beta-amino-ethyl ether)N,N'-tetraacetic acid (EGTA) (3 mM) yielded a threefold increase in cGMP in 10 min when applied to the photoreceptor surface of isolated retinas. However, when the vitreal surfaces of everted eye cups were suprafused with calcium-chelated medium for 10 min, a threefold increase in cGMP required 20 mM EGTA, 3 mM EGTA being ineffective. In another study, vitreal surfaces of eye cups or photoreceptor surfaces of retinas were suprafused with physiological saline containing 3 mM 3-isobutyl-1-methylxanthine (IBMX) in order to alter cAMP retinal levels. In both cases, eightfold increases in cAMP were measured in the inner nuclear and inner plexiform layers, while three- to fourfold increases occurred in the outer nuclear and outer plexiform layers. This agent also caused a large increase in cAMP in the pigment epithelium.