Kinetic measurements of the bacteriostatic, bactericidal and bacteriolytic activities of six model antibiotics (ampicillin, erythromycin, nalidixic acid, polymyxin B, tetracycline, and trimethoprim) against Escherichia coli as target bacteria were performed by bioluminescence, fluorescence, and optical density based real-time assay. Additionally, plate counting was used as a control measurement. The gfp and insect luciferase (lucFF) genes were cloned into cells used for measurements to enable fluoro-luminometric detection. Bacteria were exposed to antibiotics for 10 h, and the effects of antimicrobial agents were established. Inhibitory concentration of 50% (IC(50)), minimum bactericidal concentration (MBC), and bactericidal concentration of 50% (BC(50)) of each antibiotic were calculated from these procedures. Bacteriostatic, bactericidal or bacteriolytic actions of each antibiotic, as well the time interval from exposure to visible effect, were readily observed from kinetic data. No significant differences were observed between data obtained with the different methods employed. Ampicillin and polymyxin B were clearly bacteriolytic, nalidixic acid and tetracycline showed bactericidal effects, and erythromycin and trimethoprim were bacteriostatic drugs. The assay has the advantage of speed and accurately discerns between lytic, cidal and static compounds. Thus, this reliable and fully automated novel kinetic assay with high sample capacity offers new possibilities for real-time detection, making it suitable for diverse high throughput screening (HTS) applications.