A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax-DEN2) in comparison to that of YF vaccine (YF-VAX). Forty-two healthy YF naïve adults randomly received a single dose of either ChimeriVax-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAX by the subcutaneous route (SC). To determine the effect of YF preimmunity on the ChimeriVax-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVax-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3 and 4 were observed in YF naïve subjects inoculated with either ChimeriVax-DEN2 or YF-VAX. In contrast, 100% of YF immune subjects inoculated with ChimeriVax-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2 and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that (1) the safety and immunogenicity profile of the ChimeriVax-DEN2 vaccine is consistent with that of YF-VAX, and (2) preimmunity to YF virus does not interfere with ChimeriVax-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.