In vivo studies show that beta3-integrin-mediated focal adhesion formation (FAF) causes recruitment of nonreceptor tyrosine kinases to the cytoskeleton in pressure-overloaded myocardium. To define the mechanism of beta3-integrin-mediated signaling, we developed a cell culture model (adult feline cardiocytes embedded in a 3-dimensional matrix of native type 1 collagen, fibronectin, and vitronectin) wherein beta3-integrin-mediated focal adhesion kinase occurs. Focal adhesion kinase was analyzed immunocytochemically using confocal microscopy. Initial studies suggested that cardiocytes cultured in a 3-dimensional matrix formed focal adhesions consisting of both beta3-integrin and the muscle-specific isoform, beta1-integrin (beta1D). The focal adhesions were associated with focal adhesion kinase on both costameres and intercalated disks. To determine the cause of beta1D-integrin-mediated focal adhesion kinase in this model, time course studies were done. Beta3-integrin-mediated focal adhesion kinase occurred within 30 minutes after embedding cardiocytes and persisted for >24 hours, whereas beta1D-integrin-mediated focal adhesion kinase was present from the outset. Because confocal microscopy showed that laminin was present on the surface of freshly isolated cardiocytes, we hypothesized that this was causative of beta1D-integrin-mediated focal adhesion kinase. Freshly isolated cardiocytes washed with acidic medium (2 minutes, pH 3.0) to remove laminin and then embedded in a 3-dimensional matrix showed complete absence of beta1D-integrin-mediated focal adhesion kinase, but beta3-integrin-mediated focal adhesion kinase occurred with a time course similar to that seen in cultured, unwashed cardiocytes. Acid washing did not alter the binding ability of beta1D-integrin, because acid-washed cardiocytes in the presence of laminin showed beta1D-integrin-mediated focal adhesion kinase. Thus, cardiocytes embedded in a 3-dimensional matrix show beta3-integrin-mediated focal adhesion kinase and provide an in vitro model to study beta3-integrin-mediated signaling in response to hemodynamic cardiac loading.