The stability of five major beta-lactam antibiotics (amoxicillin, ampicillin, cloxacillin, oxacillin, and penicillin G) in fortified milk and in milk extracts prepared for LC-ESIMS/MS analysis was studied at varying cold temperatures (4, -20, and -76 degrees C). Storage of milk samples at 4 degrees C resulted in measurable losses of all beta-lactams after 6 days (>50% in most cases). Slow degradation of penicillin G, cloxacillin, and oxacillin was observed in milk stored at -20 degrees C, but no losses were recorded at -76 degrees C over 4 weeks. All antibiotics showed good stability at all temperature tested in milk extracts prepared for LC-ESIMS/MS analysis. The results of this study emphasize adherence to adequate sample handling and storage protocols as to reflect residue levels at the time of sample submission.