Rocio encephalitis is an epidemic flaviviral infection of man first described in Sāo Paulo State, Brazil, in 1975. The virus has been isolated from arthropods (Psorophora ferox) collected in nature only once, and the ecology of the viral transmission cycle remains largely unknown. Rocio virus produces high level viremias (106.0 to 108.0 Vero cell plaqueforming units (PFU)/ml at 48–72 hours postinfection) in young chicks following subcutaneous inoculation of virus or the bite of a single infected mosquito. We evaluated the susceptibility of a variety of mosquito species and strains to infection per os, measured virus infection and transmission rates, determined the virus content of infected mosquitoes, and obtained information on the growth pattern of Rocio virus in some of the species and strains. On the basis of these data, we classified the mosquitoes tested according to vector potential by assigning them to one of three categories. Culex tarsalis from Arizona and Cx. p. pipiens from Illinois are relatively efficient experimental vectors. Both species were readily infected by feeding on infected chicks, and high proportions (92% and 71%, respectively) were able to transmit virus on the 20th day of extrinsic incubation. Tennessee Cx. pipiens subspecies and Argentina Cx. p. quinquefasciatus are moderately efficient experimental vectors. Both strains were readily infected (94% to 98%); however, transmission rates were low (36% and 23%, respectively). Louisiana Ps. ferox, and Cx. nigripalpus and Cx. (Melanoconion) opisthopus from Florida are relatively inefficient experimental vectors. Either infection rates were low or virus did not grow to high titer in those individuals that became infected, or both. These studies provide support for the hypothesis that Rocio virus is a mosquito-borne arbovirus and illustrate the potential for transmission of the virus by two prevalent Culex species, should the agent be introduced into North America. The role of Ps. ferox as a natural vector in Brazil appears less likely on the basis of our findings, but there is a need to investigate the vector efficiency of Brazilian strains of this species.