Abstract—Cardiac fibroblasts (CFs) produce extracellular matrix proteins and participate in the remodeling of the heart. It is unknown if brain natriuretic peptide (BNP) is synthesized by CFs and if BNP participates in the regulation of extracellular matrix turnover. In this study, we examined the production of BNP in adult canine CFs and the role of BNP and its signaling system on collagen synthesis and on the activation of matrix metalloproteinases (MMPs). BNP mRNA was detected in CFs, and a specific radioimmunoassay demonstrated that BNP1-32was secreted into the media at a rate of 11.2±1.0 pg/105cells per 48 hours (mean±SEM). The amount of BNP secretion was significantly (P<0.01) augmented by 10−7mol/L tumor necrosis factor-&agr; in a time-dependent manner. BNP significantly (P<0.01) inhibited de novo collagen synthesis as assessed by [3H]proline incorporation, whereas zymographic MMP-2 (gelatinase) abundance was significantly (P<0.05) stimulated by BNP between 10−7and 10−6mol/L. In addition, protein expression of MMP-1, -2, and -3 and membranous type-1 MMP was significantly increased by 10−6mol/L BNP. The cGMP analogue 8-bromo-cGMP (10−4mol/L) mimicked the BNP effect, whereas inhibition of protein kinase G by KT5823 (10−6mol/L) significantly (P<0.05) attenuated BNP-induced zymographic MMP-2 abundance. In summary, this study reports that BNP is present in cultured CFs and that BNP decreases collagen synthesis and increases MMPs via cGMP–protein kinase G signaling. These in vitro findings support a role for BNP as a regulator of myocardial structure via control of cardiac fibroblast function.