The feasibility of a purified, inactivated dengue (DEN) vaccine made in Vero cells was explored. A DEN-2 virus candidate was chosen for production of a monotypic, purified, inactivated vaccine (PIV). Virus was harvested from roller bottle culture supernatants, concentrated, and purified on sucrose gradients. The purified virus was inactivated with 0.05% formalin at 22°C. After inactivation, the virus retained its antigenicity and was immunogenic in mice and rhesus monkeys, in which it elicited high titers of DEN-2 virus-neutralizing antibody. Mice were completely protected against challenge with live, virulent virus after receiving two 0.15-µg doses of PIV. Monkeys vaccinated with three doses ranging as low as 0.25 µg demonstrated complete absence or a significant reduction in the number of days of viremia after challenge with homologous virus. These results warrant further testing and development of PIVs for other DEN virus serotypes.