PURPOSE: To determine whether rose bengal and lissamine green affect polymerase chain reaction (PCR) detection of herpes simplex virus (HSV). DESIGN: Laboratory investigation. METHODS: Diagnostic corneal scrapings were evaluated for PCR inhibitory activity. Dacron swabs inoculated with rose bengal and lissamine green were processed as clinical samples, inoculated with control HSV, varicella zoster (VZV), cytomegalovirus (CMV), and toxoplasma DNA and prepared for PCR. The effects of calcium alginate and cotton swabs were also evaluated. RESULTS: Rose bengal, lissamine green, and calcium alginate not only inhibit PCR detection of HSV DNA, but also detection of VZV, CMV, and toxoplasma DNA. This inhibition could be overcome by serial dilution and by DNA purification of the sample before PCR. CONCLUSIONS: Rose bengal, lissamine green, and calcium alginate can inhibit PCR detection of HSV DNA. Clinical scrapings to be sent for PCR diagnostic testing should be taken before instillation of rose bengal or lissamine green.