Factor VIIIa is a heterotrimer of A1,A2 and A3-C1-C2 subunits which is labile due to a relatively weak affinity interaction between the A2 subunit and the Me(2+)-linked A1/A3-C1-C2 dimer. Previously we speculated that the acidic region at the COOH terminus of the A1 subunit was involved with the A2 subunit retention. This region, delineated by factor VIII residues 337-372, was chemically synthesized. Both the peptide, designated FVIII337-372, and an IgG fraction prepared from rabbit anti-FVIII337-372 antiserum inhibited the reconstitution of factor VIIIa from A1/A3-C1-C2 dimer plus A2 subunit. A primary component of the inhibitory activity of the peptide was attributed to its acidic nature based upon similar inhibition of factor VIIIa reconstitution using a synthetic polymer of aspartic acid. Trypsin cleaved the peptide at Arg359 and the resultant two fragments were isolated. Inhibitory activity was associated with the NH2-terminal fragment which contained 10 of the 13 acidic residues present in the original peptide. The fluorescence of a dansylated FVIII337-372 was enhanced 2-fold by A2 subunit and this effect was reversed by addition of excess unmodified peptide. The inhibitory activity of FVIII337-372 was attenuated by the presence of Ca2+. Ca2+ also inhibited the reconstitution of factor VIIIa in the absence of peptide and increased the rate and extent of factor VIIIa decay, suggesting that Ca2+ effectively shielded charges important for the intersubunit interactions. The above results support a role for this acidic region in the association of A2 subunit with A1/A3-C1-C2 dimer.