Purified human peripheral blood T lymphocytes were shown to adhere to growth substrata coated with purified human plasma fibronectin (pFn) and its Mr 120,000-140,000 proteolytic fragments containing the cell-binding site. In contrast, significant binding to laminin- or type I collagen-coated surfaces could not be demonstrated. Binding of T cells to pFn could be inhibited by the synthetic peptide Arg-Gly-Asp-Ser. Activation of T lymphocytes with concanavalin A (Con A) and a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), led to a higher adherence to pFn than in unstimulated, resting T cells. Activation with only Con A in the presence of accessory cells also promoted binding. Increased adherence of T cells to pFn could be demonstrated as early as 2 h after the onset of stimulation and reached its maximum in 2-3 days. Furthermore, activated but not resting T cells actively spread on pFn-coated surfaces and displayed an altered F-actin organization. In an overlay assay of electrophoretically separated polypeptides of activated T lymphocytes, pFn bound to a high molecular weight polypeptide of Mr 190,000, suggesting that the cells bind to pFn via a receptor-like molecule. Thus, adhesion ot pFn may be a two-stage process. At the first stage cells bind to Fn. Activated T cells proceed to the second stage, where cells begin to spread on pFn. This may be due to an altered relationship between Fn receptors and microfilaments.