Despite numerous reports suggesting that ␤ 1 integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that ␤ 1 integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the ␤ 1 integrin subunit becomes hyposialylated, suggesting that the ␤ 1 integrin is a substrate for this enzyme. The expression of hyposialylated ␤ 1 integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified ␣ 5 ␤ 1 integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated ␤ 1 integrins are more adhesive to fibronectin, although desialylation of ␣ 5 subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the ␤ 1 integrin family of cell adhesion receptors.