A total of 84 virus strains was obtained from 16,374 male and female sand flies (Phlebotomus perniciosus and P. perfiliewi) collected in two localities of Tuscany region in Italy between 1980 and 1985. Thirty-seven (44%) were identified as Toscana virus (family Bunyaviridae, genus Phlebovirus) and 47 (56%) as a new member of the Phlebotomus fever serogroup, Arbia virus. The characteristics of this new serotype are described. The overall virus isolation rate from sand flies was 0.5 per 100 insects processed. Virus isolation rates for both viruses were similar in different years and in the two localities, suggesting that the two virus types were active in the sand fly population simultaneously. Each year, the largest number of isolates were obtained during July, corresponding to the period of maximal sand fly population density. Both viruses were repeatedly isolated from male sand flies, suggesting transovarial transmission in nature. Serologic data showed no evidence of infection among domestic and wild animals. However, a strain of Toscana virus was isolated from the brain of a bat (Pipistrellus kuhli), indicating a possible involvement of this species in the ecology of the virus. Serologic tests did not provide definitive evidence for human infection by Arbia virus.