Two-dimensional electrophoretograms of serum proteins from ampicillin-treated patients were analyzed-by immunoblotting with an antiserum specific for penicilloyl groups. As expected, human serum albumin (HSA) was the main ampicilloylated serum component. Transferrin main form II was found to be the second most important component as regards immunoblotting intensity. Immunoreactive spots were present on the acidic side of the transferrin isoelectric series, suggesting a modification mechanism similar to that observed in HSA, i.e., acylation of basic amino acid residues. Several additional ampicilloylated spots were detected but could not be assigned. Their electrophoretic parameters were determined using internal standards. This is the first description of serum proteins other than HSA being modified by ampicillin in the course of routine therapeutic treatment.