Heart failure is the leading cause of combined morbidity and mortality in the United States. It remains an incurable disease process with an estimated two-year mortality of 30-50% for the patients with advanced disease. Although advances in the treatment for failing heart have been made, our understanding of the molecular mechanism leading to heart failure is still limited. The Schwartz lab showed that proliferative fibroblasts derived from monocytic, blood-borne precursor cells play a critical role in fibrosis in a murine ischaemic/reperfusion cardiomyopathy (I/RC) model. We showed that down regulation of Rho Kinase-1 (ROCK1) blocked the movement of monocytes from the coronary blood vessels into the heart to form myofibroblasts that cause fibrosis. Our Rho Kinase-1 (ROCK-1) null mice did not develop cardiac fibrosis and dysfunction, identifying a powerful rationale to develop ROCK inhibitors for the treatment of heart disease. We discovered an inhibitory polypeptide, peptide 25, which blocked ROCK1 activity in the presence of 1mM ATP. The peptide 25 binding epitope on ROCK1 was mapped using chemical cross-linking to the Activation Loop, a novel locus to direct a new class of inhibitory drugs. We synthesized alanine scanning and truncation mutants from peptide 25 to find which amino acids are critical for binding to the ROCK1 activation loop. Molecular modeling was used to identify the structural interactions of the peptide with the activation loop on the catalytic domain.