An automated immunosensor for the rapid and sensitive analysis of penicillin type beta-lactam antibiotics has been developed and optimized. An immunogen was prepared by coupling the common structure of the penicillanic beta-lactam antibiotics, i.e., 6-aminopenicillanic acid to keyhole limpet hemocyanin. Polyclonal antibodies raised in rabbits after immunization with this conjugate have been applied for the development of a competitive fluoroimmunoassay (FIA), using a novel fluorescent penicillin {[2S,5R,6R]-3,3-dimethyl-7-oxo-6-[(pyren-1ylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxilic acid, PAAP} as the tracer and penicillin G as the reference antibiotic. Protein A/G covalently bound to an azlactone-activated polymeric support was used for the orientated capture of the antibody-antigen immunocomplexes. Upon desorption from the immunosupport, the emission signal generated by the PAAP-Ab complexes is related to the antibiotic concentration in the sample. The 50% binding inhibition concentration of penicillin G standard curves was at 30 ng mL(-)(1) with a detection limit (10% binding inhibition) of 2.4 ng mL(-)(1) and a dynamic range from 6.0 to 191 ng mL(-)(1) (20-80% binding inhibition) penicillin G. The generic nature of the antiserum was shown by good relative cross-reactivities with penicillin type beta-lactam antibiotics such as amoxicillin (50%), ampicillin (47%), and penicillin V (145%) and a lower response to the isoxazolyl penicillins such as oxacillin, cloxacillin, and dicloxacillin. No cross-reactivity was obtained for cephalosporin type beta-lactam antibiotics (cephapirin), cloramphenicol, or fluoroquinolones (enrofloxacin and ciprofloxacin). The total analysis time was 23 min per determination, and the immunoreactor could be reused for more than 200 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of penicillin G and amoxicillin in spiked influent and effluent sewage treatment plant water samples with excellent recoveries (mean values for penicillin G and amoxicillin, 99 and 105%, respectively). Results displayed by comparative analysis of the immunosensor with a chromatographic procedure for penicillins showed excellent agreement between both methods.