Studies on bacterial metabolism during the postantibiotic effect (PAE) period are limited but might provide insight into the nature of the PAE. We evaluated the rate of DNA synthesis in bacteria during the PAE period after a 1-h exposure of organisms in the logarithmic growth phase to various antibiotics. Staphylococcus aureus ATCC 25923 was exposed to vancomycin, dicloxacillin, rifampin, and ciprofloxacin; Escherichia coli ATCC 25922 was exposed to gentamicin, tobramycin, rifampin, imipenem, and ciprofloxacin; and Pseudomonas aeruginosa ATCC 25783 was exposed to imipenem, tobramycin, and ciprofloxacin. DNA synthesis was determined by measuring the rate of [3H]thymidine incorporation in S. aureus and E. coli and [3H]adenine incorporation in P. aeruginosa. DNA synthesis in S. aureus was suppressed during the PAE phase with vancomycin, dicloxacillin, and rifampin, it was suppressed in E. coli with rifampin, and it was suppressed in P. aeruginosa after exposure to tobramycin. Conversely, DNA synthesis was relatively enhanced in the gram-negative bacilli after exposure to imipenem and in all three species after exposure to ciprofloxacin. However, DNA synthesis in E. coli was only minimally affected after exposure to tobramycin and gentamicin. The differences in DNA synthesis observed after exposure to various antimicrobial agents suggest multiple mechanisms for the PAE.