The intestinal response to injury requires coordinated regulation of the tension exerted by subepithelial myofibroblasts (SEM). However, the signals governing relaxation of intestinal SEM have not been investigated. This study's aim was to test the hypothesis that signal transduction pathways initiated by C-type natriuretic peptide (CNP) induce intestinal SEM relaxation. We directly quantified the effects of CNP on the isometric tension exerted by cultured human colonic SEM. We also measured the effects of CNP on cGMP content, myosin regulatory light chain (MLC) phosphorylation, and cytosolic [Ca²⁺]. CNP induced relaxation of SEM within 10 seconds. By 10 minutes relaxation reached a plateau that was sustained for two hours. CNP-induced relaxation was saturable with a maximal decrease in tension (51.7 [plusmn]] 3.8 dynes) observed at 250 nM. SEM relaxation in response to CNP constituted approximately 23% of total basal tension. CNP increased intracellular cGMP content and reduced MLC phosphorylation. The effects of CNP on cGMP and MLC exhibited the same dose dependence as CNP-induced relaxation. MLC phosphorylation decreased within 2 minutes of CNP exposure, and was sustained for at least 45 minutes. CNP also stimulated a large transient increase in cytosolic [Ca²⁺] that occurred within 30 seconds and was nearly complete by one minute. We also observed that calyculin-A, a potent inhibitor of MLC phosphatase, completely abolished the reduction in MLC phosphorylation induced by CNP. These results suggest that CNP induces intestinal SEM relaxation through cGMP-associated reductions in MLC phosphorylation. Moreover, these findings raise the possibility that CNP may play a role in intestinal wound healing.