Cardiac fibroblasts account for up to two-thirds of the total number of cells in the normal heart and are responsible for extracellular matrix homeostasis. In vitro type I collagen, the predominant myocardial collagen stimulates proteolytic activation of constitutively secreted proMMP-2 at the cell membrane and requires formation of a ternary complex with membrane-type metalloproteinase (MT1-MMP) and TIMP-2. Following myocardial infarction (MI) normally quiescent fibroblasts initiate a wound healing response by transforming into a proliferative and invasive myofibroblast phenotype. Myocardial oxygen deprivation is an inevitable consequence of MI, therefore this reparative event occurs under chronically hypoxic conditions. The aim of this study was to investigate the effect of hypoxia on fibroblast migration and invasion, and to investigate whether modulation of MMP-2 activation was involved. Human cardiac myofibroblasts were cultured from biopsies of right atrial appendage from patients undergoing coronary artery bypass surgery. All experiments were performed on cells from at least 5 different patients in medium supplemented with 50 mg/ml Type I rat tail collagen under conditions of normoxia (21% O2) and hypoxia (1% O2) for 48 h. Invasion assays were performed using a modified Boyden chamber technique with Matrigel coated membranes and 2.5% FCS as chemoattractant. Invaded cells were quantified by counting H & E stained cell nuclei on the underside of each membrane under light microscopy. Migration was quantified using a scratch wound assay by making a uniform linear wound in a confluent monolayer of myofibroblasts. Quantification was performed from digital images of the wound before and after incubation. Cell supernatants were collected at the end of all experiments, and analysed by gelatin zymography to quantify pro and active MMP-2 secretion. 2.5% FCS significantly stimulated myofibroblast invasion, which did not occur in the absence of chemoattractant. Hypoxia (1% O2) significantly attenuated invasion by ~50% (P<0.05, paired t-test, n=7). The inhibition was accompanied by reduced MMP-2 activation in invasion assay supernatants of ~50% (P<0.05, paired ratio t-test, n=7). In contrast, in the wound scratch assays, despite observing a significant inhibition of MMP-2 activation (P<0.01, n=5), hypoxia had no effect on cell migration (P=NS, n=5). In conclusion, the lack of effect of hypoxia on migration suggests that inhibition of invasion is mediated by inhibition of MMP-2 activation but not by modulation of fibroblast motility. Decreased activation of MMP-2 following hypoxia potentially impairs early, post-MI remodelling. Determining the exact mechanism by which this occurs may provide a target for therapeutic modulation.