The effect of concanavalin A (Con A) on the adhesion of 8-day-old chick embryo fibroblasts (CEFs) to fibronectin (FN) and laminin (LM) was studied. Con A was shown to inhibit the spreading of CEF on a LM substrate. In contrast, no inhibition of CEF spreading on the FN substrate could be detected when the quantity of FN coated varied from 0.5 to 4 pmoles. The effect induced by Con A was specific, since it was abolished by 100 mM alpha-methylmannopyranoside. The inhibition of CEF spreading was only observed when the lectin was added during the 20 min following cell plating. In addition, the effect of Con A on CEF spreading on the LM substrate was shown to be dependent upon its presence at the cell surface, since under conditions which accelerate the uptake of the lectin, the effect on cell spreading is no longer detectable. Furthermore, the number of CEFs attached to LM was not modified by the lectin. The molecular weight of the isolated Con A binding sites revealed glycoproteins ranging from 30,000 to 72,000. On the other hand, these Con A binding sites did not interact with LM-Sepharose. Only a protein with a molecular weight of 68,000 which did not express affinity for Con A bound tightly to the LM-Sepharose. These data suggested that cell surface Con A binding sites do not interfere with the initial step of CEF adhesion to LM but play a key role during their spreading on this glycoprotein.