Purpose: C-type natriuretic peptide (CNP)-22, produced in the endothelium and kidney, is a potent antifibrotic factor functioning through the activation of guanylyl cyclase receptor B (GC-B) and its second messenger, cyclic GMP (cGMP).Evidence has suggested that CNP-22 serves as a cardiorenal protective peptide, activated by humoral mechanisms in the setting of injury, to suppress profibrotic processes and preserve function. CNP is produced as proCNP which undergoes processing by furin and an unknown enzyme to yield NT-proCNP,CNP-53,NT-CNP-53 and the active form CNP-22.To date, it remains unclear whether different CNP molecular forms (MFs) are present in human plasma and urine or if all of these CNP MFs have the ability to activate GC-B or cGMP. We hypothesized that the MFs, CNP-53, NT-CNP-53 and CNP-22 will be detectable in normal human plasma and urine. We also hypothesized that, based on their structure,only the MFs CNP-22 and CNP-53 would have the cGMP-activating actions in human fibroblasts (hFs) that would be specific for GC-B. Methods: Plasma levels and urinary excretion rate of CNP-22, CNP-53 and NT-CNP-53 in 20 normal adults (age 54±6 yrs) were measured by radioimmunoassay (RIA).Cultured hFs, which highly expresses GC-B, were stimulated with 10-8M proCNP, NT-proCNP,CNP-53,NT-CNP-53 or CNP-22 and cGMP was measured by RIA. HEK 293 cells expressing either GC-A or GC-B were stimulated with CNP-53 or CNP-22 (10-8M) and cGMP was measured to determine receptor specificity.Data are mean±SE;*p<0.05. Results: In human plasma, CNP-53 and CNP-22 levels were detectable and similar in concentration, with both of these forms being significantly greater than plasma NT-CNP-53 (11±5 and 12±3 vs 6±3*pg/ml). These MFs were also in the urine and the urinary excretion rate of CNP-53 was significantly higher than both NT-CNP-53 and CNP-22 (4670±974 vs 1137±140 and 417±28*pg/h). In hFs, CNP-22 and CNP-53 activated cGMP, however CNP-22 was twice as potent as CNP-53 (0.31±0.01 vs 0.17±0.03* pmol/ml). The cGMP responses to proCNP, NT-proCNP and NT-CNP53 were similar to untreated cells at a level of 0.001±0.001 pmol/ml.HEK293 cell studies revealed that CNP-53 and CNP-22 were specific agonists of GC-B and not GC-A. Conclusion: We demonstrate for the first time that CNP MFs are detectable at various levels in human plasma and urine, as well as having differential cGMP activating properties in hFs,with only CNP-22 and CNP-53 having selective GC-B/cGMP activity (CNP-22>-53). Future studies are needed to investigate the biological role of CNP MFs as potential biomarkers for cardiorenal injury and novel antifibrotic drugs.