A microbiological screening method (three-plate) for the detection of the antimicrobial growth promoters tylosin, spiramycin, virginiamycin, zinc bacitracin, and avoparcin in animal feed has been developed and validated successfully. A collaborative study involving 18 laboratories receiving 172 samples was carried out to verify the performance characteristics. The detection level for tylosin/virginiamycin/spiramycin, expressed in microbiological activity, was 1 mg kg-1 (false-positives, 2%; false-negatives, 3, 0, and 6%, respectively). Avoparcin could be detected at 1 mg kg-1 in feed in general (false-positives, 2%; false-negatives, 0%). However, in calf feed the sensitivity was lower. The percentages of false-negatives were found to be 12%, 7%, and 0% at 1, 3, and 5 mg kg-1, respectively (false-positives, 4%). The limit of detection for zinc bacitracin was 3-5 mg kg-1 (false-positives, 5-10%; false-negatives, 77% at 1 mg kg-1, 45% at 2 mg kg-1, 12% at 3 mg kg-1, and 4% at 5 mg kg-1). The method allowed for a distinction to be made between the groups of antibiotics: avoparcin/zinc bacitracin versus tylosin/virginiamycin/spiramycin. This definitely gives added value to the method in the framework of a follow-up of positive screening results by post-screening and confirmatory analysis.