The α6A and α6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the α6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of α6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the α6 precursor. Mutations were introduced in the codons for the R⁸⁷⁶KKR⁸⁷⁹, E⁸⁸³K⁸⁸⁴, R⁸⁹⁰K⁸⁹¹ and R⁸⁹⁸K⁸⁹⁹ sequences, the potential proteolytic cleavage sites, and wild-type and mutant α6A cDNAs were transfected into K562 cells. The mutant α6A integrin subunits were expressed in association with endogenous β1 at levels comparable to that of wild-type α6Aβ1. A single α6 polypeptide chain (150 kDa) was precipitated from transfectants expressing α6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded α6A subunits that were cleaved once into a heavy and a light chain, whereas α6A subunits with mutations in one of the two RK sequences were, like wild-type α6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of α6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two α6A light chains from platelets demonstrated that cleavage occurs after Arg⁸⁷⁹ and Lys⁸⁸⁴. Because α6^RKKG, α6^GKKR and α6^RGGR subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. α6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys⁸⁸⁴, were partly cleaved, whereas α6A mutants with the RKKR sequence shifted to other positions in the α6A subunit, including one in which it was shifted two residues farther than the EK cleavage site, were not cleaved. In addition, α6A mutants with an α5-like cleavage site, i.e. arginine, lysine and histidine residues at positions -1, -2 and -6, were not cleaved. Thus both an intact RKKR sequence and its proper position are essential. After activation by the anti-β1 stimulatory monoclonal antibody TS2/16, both cleaved and uncleaved α6Aβ1 integrins bound to laminin-1. The phorbol ester PMA, which activates cleaved wild-type and mutant α6Aβ1, did not activate uncleaved α6Aβ1. Thus uncleaved α6Aβ1 is capable of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of α6 is required to generate a proper conformation that enables the affinity modulation of the α6Aβ1 receptor by PMA.