AbstractThe laboratory preparation of chemically pure, ³H-labelled β-propionylampicillin suitable for studies of the mode of action of β-lactam antibiotics has been investigated. The chemical purity of the labelled antibiotic is a crucial factor for the quantitative determination of penicillin-binding proteins. Ampicillin (D-α-aminobenzylpenicillin) was N-[³H]propionylated by N-succinimidyl [³H]propionate, the labelled reaction products were analyzed by thin-layer chromatography followed by fluorography. The synthesis and the purification of the resulting N-[³H]propionylampicillin have been optimized, the best conditions for the acylation reaction were at pH 8.25 for 2 h, giving both a high yield of the labelled antibiotic and minimal amounts of unwanted by-products. The reaction mixture was fractionated by a two-step procedure, the purified radioactive antibiotic did not contain any residual reactant, especially free ampicillin, or degradation products such as D-α.-[³H]propionamidobenzylpenicilloic acid. This improved procedure allows an easy, fast and rather inexpensive preparation of a good quality radiolabeled antibiotic for studies of penicillin-interacting proteins.