A rapid method was developed for specific detection of microbial 1-lactamases which uses ampicillin and cephalexin as substrates. The end products (open 3-lactam ring forms) generated after separately incubating either substrate with 3-lactamase-producing organisms initially were separated from the unhydrolyzed substrates by high-voltage electrophoresis at pH 2.1. The end products of both antibiotics were highly fluorescent and could be analyzed visually and semiquantitatively under a long-wave UV lamp. Application of 5 ,ul of the same incubation mixture onto filter paper without subsequent electrophoretic separation also resulted in development of fluorescence after brief heating at 120°C for 5 min. This spot test differentiates penicillinase activity from cephalosporinase activity and distinguishes between 3-lactamase and acylase activities, since the end products of acylase [the common side chain, D(-)-a-aminophenylacetic acid, and the intact 3-lactam nuclei, 6-aminopenicillanic acid and 7-aminodeacetoxycephalosporanic acid] are not fluorescent. This method was relatively rapid, inexpensive, and more sensitive than the chromogenic cephalosporin (nitrocefin) method when 21 strains of 7 gram-positive species and 77 strains of 29 gramnegative species of bacteria were tested. P-lactamases which hydrolyze the amide bonds of the ,Blactam ring of sensitive penicillins and cephalosporins (Fig. 1 and 2) are widely distributed among microorganisms (14) and play ah important role in microbial resistance to f-lactam antibiotics. Chemical methods used for detecting microbial * Corresponding author.