A new virus, SP An 71686, was isolated from sentinel mice exposed in a forest area in Iguape county, Sao Paulo state, Brazil, in 1979. The results suggest [hemagglutination inhibition (HI), complement fixation, neutralization, and ELISA] that SP An 71686 virus is a new arbovirus and that it demonstrates some cross-reactivity with other members of the family Flaviviridae, but can be differentiated from them. Although there is an intensive circulation of several arboviruses in the area, the only diagnosed cases of human disease were caused by Rocio virus during and after the epidemic of encephalitis that occurred in 1975-1977, one case of febrile illness by Caraparu virus in 1983, and by subtype IF of Venezuelan equine encephalitis virus in soldiers during jungle survival training in 1990. Wild animals had a prevalence of SP An 71686 HI monotype antibodies: 46% of birds captured in 1990, 40% in 1991 and 19.5% in 1992. These results suggested that wild birds may play a role in the virus transmission cycle. Mammals (rodents and marsupials) must also be considered potential hosts. However, the virus reservoir-vector relationships need further studies which would help to clarify the ecology of this virus.