The antibiotic bacerial inactivity of N-acetylcysteine (NAC) and its interaction with penicillin and aminocyclitol antibiotics was evaluated. NAC inhibited growth of both gram-negative and gram-positive bacteria. Strains of Pseudomonas aeruginosa were more susceptible than other microorgainsms tested. P. aeruginosa strains were inhibited synergistically by NAC and carbenicillin or ticarcillin. However, NAC antagonized the activity of gentamicin and tobramycin. These findings have implications for the combined clinical use of NAC and aerosolized antibiotics and are also important for the processing of sputum specimens in the microbiology laboratory.