Background/aims: Pseudomonas aeruginosa is a major cause of severe bacterial keratitis and remains a difficult clinical entity to treat successfully with the current arsenal of antimicrobial agents. Defensins are small cationic peptides with broad in vitro antimicrobial activity and are potential ocular therapeutic agents. The authors characterised the in vitro activity of defensins NP-1 and NP-3a against P aeruginosa in the presence of human tears. Methods: A clinical Pseudomonas isolate was grown to mid-log phase, and 1×106 colony forming units were exposed to the peptides (200 μg/ml) for up to 2 hours in the presence of varying concentrations (10–70%) of human tears. Results: For both peptides in the presence of 10% tears, >3 log units of killing was achieved within 30 minutes. In 70% tears, NP-1 produced >1 log unit of killing at 2 hours, indicating that, although reduced, its activity remained significant. In 20% tears, NP-3a demonstrated 2 log units of killing at 2 hours; however, the antimicrobial activity of this defensin was completely inhibited in the presence of 70% tears. Conclusion: These in vitro data suggest that while the microbicidal activity of some defensins may be diminished at the ocular surface in vivo, significant activity is still possible with certain peptides.