The stability of Dryvax vaccine was examined to determine whether diluted vaccine could be used over an extended period, thereby increasing the number of available doses. Stability parameters that we evaluated include dilution (undiluted, diluted 1:5, and diluted 1:10), diluent (original Dryvax diluent, new Dryvax diluent, and phosphate-buffered saline), and temperature (refrigerator temperature and room temperature). Storage of reconstituted Dryvax vaccine at room temperature, regardless of dilution or diluent, resulted in a significantly greater decrease in titer than did storage at refrigerator temperature. At refrigerator temperature, the decrease in vaccinia-vaccine titer during the first year was not significantly different between the undiluted Dryvax vaccine and either of the 2 diluted forms of Dryvax vaccine, with any of the 3 diluents.