Antibodies against the benzylpenicilloyl determinant were used to identify complexes of benzylpenicilloyl and penicillin binding protein (PBP) of several bacterial species on immunoblots. Since radioactive penicillin was not needed, this technique readily allowed in vivo labeling studies even in Escherichia coli, where the saturating concentration was around 0.6 mg/ml. The antibodies showed no substantial cross‐reactivity to other β‐lactam‐PBP complexes with the exception of 6‐aminopenicillanic acid. Surprisingly, some penicilloyl‐PBP were hardly recognized by the antiserum, wherease the others could be stained according to the amount of penicillin bound.