Tryptic digests of fragment A299-585 of penicilloylated serum albumin obtained from two penicillin-treated patients or prepared by in vitro conjugation, were analyzed by a tandem immunoaffinity reversed-phase HLPC. Determinations of benzyl penicilloyl groups (BPO) were performed on the different fractions. Three BPO containing peptides were identified by their amino acid sequence and the bound BPO were located on lysines 432, 541 and 545. Six major BPO binding sites were thus identified on the whole albumin molecule. All of them are lysine residues and correspond to a limited number of definite structures in which lysine and serine residues appear to be closely associated.