The synthesis and properties of six fluorescein-labelled penicillins are reported. The two isomers of fluoresceyl-glycyl-6-amino-penicillanic acid are probably the best compounds to use for detection of all the penicillin-binding proteins (PBPs) present in a bacterial membrane preparation. However, the derivatives of ampicillin were much more efficient against Enterobacter aerogenes PBP3. The two isomers obtained when a commercial mixture of the two isomers of carboxyfluorescein was used most often exhibited similar properties, but the Streptomyces R61 extracellular DD-peptidase was only efficiently acylated by the 5'-carboxyfluorescein derivative of glycyl-6-aminopenicillanic acid.