The first confirmed outbreak of dengue fever in Peru occurred during 1990 in Iquitos, a city of approximately 300,000 residents in the Amazon region. Because of the apparent establishment of endemic transmission of this mosquito-borne viral disease following the outbreak, epidemiologic studies were initiated in 1992. Blood specimens and data on demographic, environmental, and medical history factors were collected from volunteers in an urban sector of Iquitos, in a rural area on the outskirts of Iquitos, and in three nearby jungle communities. A follow-up blood specimen was obtained approximately one year later from a sample of subjects. Sera were tested for dengue IgG antibody by an enzyme-linked immunosorbent assay, and specificity was verified using a plaque-reduction neutralization test. Dengue antibody prevalence was 66% in the urban population, 26% in the rural population, and 32–67% in the three jungle areas. A significant association was found between age and antibody prevalence, with a steady increase in prevalence from 18% among subjects less than five years of age to greater than 90% for subjects more than 50 years old. Increased antibody prevalence also was associated with urban and jungle residence and with a piped source of household drinking water. Seroconversions were documented in four of five surveyed communities. These results indicate that dengue virus transmission continues in and around Iquitos and suggest that transmission also occurred prior to the 1990 epidemic.