The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for a-smooth muscle actin (SMA, a myofibroblast marker) were rare (0.69±0.48%) but in sclerotic valves of animals fed an atherogenic diet myofibroblasts were spatially clustered and abundant (31.2±6.3%). In cultured VIC populations from normal valves, SMA positive myofibroblasts were also spatially clustered, abundant (21% positive cells after 1 passage) and stained for collagen type I and vimentin, but not desmin. For analysis of stem cells, two color flow cytometry of isolated cells stained with Hoechst 33342 demonstrated that 0.5% of VICs were side population cells; none stained for SMA. Upon culture, sorted side population cells generated ~85% SMA positive cells, indicating that myofibroblasts originate from a rare population with stem cell characteristics. Plating cells on rigid collagen substrates enabled the formation of myofibroblasts after 5 days in culture, which was completely blocked by culture of cells on compliant collagen substrates. Isotope coded-affinity tags and mass spectrometry were used to identify differentially expressed proteins in myofibroblast differentiation of VICs. Of the 9 proteins that were identified, cofilin expression and phospho-cofilin were strongly increased by conditions favoring myofibroblast differentiation. Knockdown of cofilin with siRNA inhibited collagen gel contraction and reduced myofibroblast differentiation as assessed by SMA incorporation into stress fibers. Compared to normal valves, diseased valves showed strong immunostaining for cofilin that co-localized with SMA in clustered cells. We conclude that in VICs, cofilin is a marker for myofibroblasts in vivo and in vitro that arise from a rare population of stem cells and require a rigid matrix for formation.