The restoration of the dark state in retinal rod cells following illumination is due in part to resynthesis of cGMP. Retinal guanylyl cyclase specifically catalyzes the cyclization of GTP into cGMP in vivo. The reaction has been shown to involve the inversion of the configuration on the phosphate atom as demonstrated by conversion of the (SP) isomer of GTP alpha S to (RP)-cGMPS by guanylyl cyclase [Senter, P. D., Eckstein, F., Mülsch, A., & Böhme, E. (1983) J. Biol. Chem. 258, 6741-6745]. Since (RP-cGMPS is not a substrate for retinal phosphodiesterase, we were able to measure cyclase activity with greater reliability using this novel assay as opposed to other published procedures. This assay has allowed us to reinvestigate the effects of adenylyl nucleotides on cyclase activity and to search for selective inhibitors of the rod-specific enzyme. We have measured the cyclase activity using homogenates of rod outer segments and a reconstituted system composed of guanylyl cyclase in washed rod outer segment membranes and the purified guanylyl cyclase activating protein. Our results indicate that 100-200 microM ATP (and other adenylyl nucleotides) stimulates guanylyl cyclase activity approximately 2-fold and that the observed stimulation of enzyme activity is independent of the free calcium concentration. In contrast to other particulate guanylyl cyclases, which are synergistically stimulated by a peptide ligand and ATP, guanylyl cyclase activating protein does not potentiate the effect of ATP, suggesting that retinal guanylyl cyclase may be regulated differently. ATP changes the Vmax of retinal guanylyl cyclase without changing the Km for (SP)-GTP alpha S.(ABSTRACT TRUNCATED AT 250 WORDS)