Factor VIII, a divalent metal ion-dependent heterodimer, contains a single copper atom, but the role of this metal in the structure and function of the cofactor is unclear. Earlier results showed that the dissociated heavy and light chains of factor VIII could be recombined in the presence of Ca(II) or Mn(II) but not Cu(II) to yield functional protein [Fay, P. J. (1988) Arch. Biochem. Biophys. 262, 525-531]. Inclusion of Cu(I) or Cu(II) inhibited the Mn(II)- or Ca(II)-dependent reconstitution of factor VIII with an IC50 approximately 10 micro M. The heavy chain was the susceptible subunit with inhibition by copper ion resulting from its reduced affinity for light chain. On the other hand, Mn(II)-dependent factor VIII reconstitutions performed with Cu(II) light chain and native heavy chain occurred at an accelerated rate (approximately 10-fold) and yielded an enhanced activity ( approximately 50%), likely reflecting an increased specific activity of the heterodimer. Cu ions enhanced the activity of EDTA-treated factor VIII in the presence of Ca(II) but not in its absence, suggesting that EDTA-treated factor VIII is not equivalent to separated subunits and that copper ions are auxiliary to ions that mediate reconstitution. Conformational analyses showed that the ellipticities and extrinsic fluorescence of both subunits were differentially affected by Cu(II) and Mn(II). These structural effects were fully reversed by EDTA. The metal ions had little if any effect on the conformation of intact factor VIII or the A1/A3-C1-C2 dimer. Mn(II) and Cu(II) stabilized the factor VIII light chain, and the latter stabilized the A1 subunit derived from the heavy chain, yielding similar thermal denaturation profiles that were distinct from that observed for the Ca(II)-stabilized subunits. Thus both subunits of factor VIII bind copper ions, and the effects of this binding differ from the interactions observed with Ca(II) or Mn(II). These data support a model where copper in factor VIII likely functions to increase specific activity of the heterodimer rather than directly mediating the intersubunit interaction.