We have used recombinant or synthetic α(IIb) and β3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance, α·β heterodimerization occurred in a 1:1 stoichiometry with a weak K(D) in the micromolar range. Divalent cations were not required for this association but stabilized the α·β complex by decreasing the dissociation rate. α·β complexation was impaired by the R995A substitution or the KVGFFKR deletion in α(IIb) but not by the β3 S752P mutation. Recombinant calcium-and integrin-binding protein (CIB), an α(IIb)-specific ligand, bound to the α(IIb) cytoplasmic peptide in a Ca2+-or Mn2+-independent, one-to-one reaction with a K(D) value of 12 μM. In contrast, in vitro liquid phase binding of CID to intact α(IIb)β3 occurred preferentially with Mn2+-activated α(IIb)β3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated α(IIb)β3, suggesting that Mn2+ activation of intact α(IIb)β3 induces the exposure of a CIB-binding site, spontaneously exposed by the free α(IIb) peptide. Since CID did not stimulate PAC-1 binding to inactive α(IIb)β3 nor prevented activated α(IIb)β3 occupancy by PAC-1, we conclude that CID does not regulate α(IIb)β3 inside-out signaling, but rather is involved in an α(IIb)β3 post-receptor occupancy event.