Despite terbinafine being fungicidal against Trichophyton rubrum in standard NCCLS assays and rapidly accumulating in nails in vivo, onychomycosis patients require prolonged terbinafine treatment to be cured. To investigate this, we developed a more clinically relevant onychomycosis in vitro test model. Human nail powder inoculated with T. rubrum and incubated in liquid RPMI 1640 salt medium, which did not support growth alone, developed extensive and invasive mycelial growth. Antifungal drugs were added at different concentrations and cultures incubated for 1 to 4 weeks. Fungal survival was determined by spreading cultures on PDA plates without drug and measuring CFU after 1 to 4 weeks incubation. Drug activity was expressed as the nail minimum fungicidal concentration (Nail-MFC) required for 99.9% elimination of viable fungus. Terbinafine Nail-MFC was 4 microg/ml after 1 week exposure, decreasing to 1 microg/ml after 4 weeks exposure, much higher than MFCs < or = 0.03 microg/ml determined in standard NCCLS MIC assays. In contrast, other clinically used drugs were unable to kill T. rubrum after 4 weeks incubation in this model. Invasive mycelial growth on nail appears to protect T. rubrum from the cidal action of systemic drugs, thus providing a rationale for the long treatment periods in onychomycosis.