1. We studied the relation of nitric oxide-mediated relaxation of longitudinal muscle to changes in cyclic GMP content of the tissue in the proximal colon of rats. 2. Dimethylphenylpiperazinium (DMPP) and electrical field stimulation (EFS) induced nitric oxide-mediated relaxation of the segments with a concomitant increase in cyclic GMP content. 3. LY 83583 and methylene blue, soluble guanylyl cyclase inhibitors, significantly inhibited the stimulatory effects of DMPP and EFS on the cyclic GMP content, but did not affect the relaxant responses of the segments to DMPP and EFS. 4. Rp-8 bromo cyclic GMPS, an inhibitor of cyclic GMP-dependent protein kinase had no effect on DMPP- and EFS-induced relaxation. 5. These data strongly suggested that nitric oxide-mediated relaxation of the rat proximal colon is not associated with change in cyclic GMP content of the tissue.