The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities.