This work evaluated the stability of diluted yellow fever vaccine in order to determine conditions that maintain the minimum of 3 log10 of 17D virus per human dose as required by WHO. The vaccines were held at 0 °C or at 37 °C and were diluted either with distilled water, with 0·15 m saline or with 0·15 m PBS at pH 5·5, 7·2 and 8·0. In a next step, stabilizer substances such as gelatin and peptone were added to the vaccines. Dilution of the vaccines in distilled water maintained the virus titre for up to three hours at 37 °C and this diluent has been adopted for routine use in Brazil.