Squamous cell lines cultured in vitro provide a potential test system for the selection of analogs that have an improved therapeutic index. The growth inhibitory effects of methotrexate and the new folate analog 10-ethyl, 10-deazaaminopterin were compared in three in vitro-cultured human head and neck squamous cell carcinoma cell lines. The inhibitory concentrations of the new analog were 10- to 100-fold lower than the inhibitory concentrations of methotrexate. The sensitivity of these three head and neck squamous cell carcinoma cell lines to both drugs was essentially the same as the sensitivity of a rhabdomyosarcoma cell line known to be very sensitive to methotrexate when the cell line is grown as a xenograft in athymic nude mice. These data indicate that 10-ethyl, 10-deazaaminopterin may be a new and effective agent against head and neck squamous cell carcinoma.