Talin establishes a major link between integrins and actin filaments, and contains two distinct integrin binding sites, one, IBS1, located in the talin head domain and involved in integrin activation, and a second, IBS2, that we have mapped to helix 50 of the talin rod domain and that is essential for linking integrin beta subunits to the cytoskeleton (1 Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J Biol Chem 282, 17280-17288). Through the combined approach of mutational analysis of the beta3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies as well as site-specific antibody inhibition experiments, we provide evidence that the integrin beta3 - talin rod interaction relies on a helix - helix association between alpha-helix 50 of the talin rod domain and the membrane proximal alpha-helix of the beta3 integrin cytoplasmic tail, and that charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin beta3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the beta3 subunit cytoplasmic helix as the integrin alphaIIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the beta3 subunit following integrin activation.