Entactin, a sulfated glycoprotein of 150-kDa, is a component of the extracellular matrix that promotes the adhesion of numerous types of cells, including lymphocytes (Li and Cheung, J. Immunol. 149, 3174, 1992), prompting us to question whether developing T lymphocytes in the thymus (thymocytes) also interact with this molecule. We thus investigated the adhesion of a thymocyte-like cell line (S49.1) to entactin, as well as the adhesion and migration of primary mouse thymocytes upon entactin-coated surfaces. In dose-response and time-course experiments, a 50 micrograms/mL coating concentration of entactin and a 60-min incubation period induced a high level (approximately 65-85%) of S49.1 cell adhesion. Preincubation of the S49.1 cells in medium containing the metabolic inhibitors sodium azide or 2-deoxy-D-glucose inhibited adhesion to entactin 47.2 and 79.5%, respectively. Furthermore, performing the adhesion assay at 4 degrees C instead of at 37 degrees C inhibited S49.1 cell adhesion 27.1%. A high percentage (approximately 90-100%) of S49.1 cells also bound to the lectin concanavalin A and to fibronectin, while laminin promoted only 19.3% adhesion. Our adhesion assay (St. John et al., J. Immunol. Methods, 170, 159, 1994) was then modified to permit a comparison of S49.1 cell adhesion strength to entactin relative to the other substrates. Consequently, Concanavalin A promoted the strongest adhesion, followed by fibronectin and then entactin. In addition, high percentages (92.5, 63, and 75.9%, respectively) of primary thymocytes from 4- to 5-week-old BALB/c mice adhered to entactin, Con A, and fibronectin, while much lower levels (7.6%) of adhesion to laminin were observed. Using a capillary tube random migration assay to measure haptokinesis, entactin-, concanavalin A-, and fibronectin-coated surfaces stimulated little migration, while laminin-coated surfaces enhanced thymocyte migration extensively. Since entactin promoted thymocyte adhesion but affected migration only marginally, we suggest that this molecule may play a role in thymocyte localization during T cell development.