Extracellular matrix (ECM) proteins can influence cell migration and differentiation in a variety of cell systems. Within the thymus, these molecules are heterogeneously distributed, and their physiological role is poorly understood. This prompted us to carry out in vitro studies using the thymic nurse cell (TNC) model. We observed that fibronectin and laminin accelerate spontaneous in vitro release of thymocytes from TNC, whereas anti-ECM antibodies exhibited a blocking effect. Similar results were obtained with anti-ECM receptor reagents. Moreover, these antibodies abrogated in vitro reconstitution of TNC complexes and thymocyte adhesion to TNC-derived epithelial cultures. Our results indicate that lymphocyte traffic in TNC (comprising both entrance into and exit from the epithelial structure) is affected by interactions involving extracellular matrix ligands and receptors. In this respect, the dynamic analysis of thymic nurse cell complexes should be regarded as a relevant in vitro tool for functional studies of distinct adhesion molecules in intrathymic lymphocyte traffic.