Most agents that regulate osteoclast bone resorption exert their effects indirectly, through the osteoblast. Nitric oxide, which stimulates soluble guanylyl cyclase, has been reported to inhibit osteoclast bone resorption directly, by a cGMP-independent mechanism(1). In this report, we demonstrate that C-type natriuretic peptide (CNP), an activator of membrane-bound guanylyl cyclase, stimulates bone resorption by osteoclast-containing 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated mouse bone marrow cultures. Quantitative reverse transcription polymerase chain reaction assays and anti-CNP immunocytochemistry were used to demonstrate that CNP is expressed in mouse marrow cells cultured in the presence, but not the absence, of 1,25-(OH)2D3. mRNA for guanylyl cyclase type B, the receptor for CNP, was expressed in cultures independent of 1,25-(OH)2D3. CNP (1 and 10 μM) elevated cGMP production in marrow cultures to 350 and 870%, respectively, of control values. 10 μM CNP increased osteoclast bone resorptive activity, measured by the resorption area on whale dentine wafers, or by the NH4Cl-inhibitable release of 3Hproline from radiolabeled bone chips, to 214 and 557% of control, respectively, without affecting osteoclast formation. Bone resorption by the marrow cultures was inhibited by 7F9.1, a monoclonal antibody raised against CNP, but not by control antibodies. These results indicate that CNP is a potent activator of osteoclast activity and may be a novel local regulator of bone remodeling.