A total of 269 virus strains was obtained from both male and female phlebotomine sandflies collected at two localities in Panama between 1969 and 1971. These isolates represented nine different virus types (Changuinola, VSV-Indiana, Punta Toro-CoAr 3319, Chagres, VP-175A, VP-437R, VP-161A, VP-488A and VP-118D) as well as several unidentified agents. Five of the virus types are new. Changuinola and Punta Toro-CoAr 3319 were the most frequently encountered agents and represented 52% and 19% of the isolates, respectively. Of man-biting sandflies processed by species, highest overall isolation rates were obtained from Lutzomyia trapidoi. Analysis of seasonal distribution of virus isolates indicated that activity of Changuinola, Chagres. VP-175 and VP-437R was continuous, while isolations of VSV-Indiana and Punta Toro-CoAr 3319 were intermittent. Seventeen isolates, representing six different virus types, were obtained from male sandflies. Isolation rates by sex for three of the virus types were similar in collections yielding comparable numbers of male and female insects. The frequency of virus isolations from male sandflies suggests that transovarial transmission of several of these agents occurs in nature. Neutralization tests on sera from inhabitants of seven rural Panamanian communities demonstrated a significant amount of human infection with VSV-Indiana. Punta Toro and VP-437R. Infections of caged sentinel animals, exposed at one of the collecting sites, were observed with VSV-Indiana, Chagres, VP-175A and VP-437R. The study demonstrated superiority of the Vero cell culture system over newborn mice for primary isolation of sandfly virus isolates. By using infected Vero cells as complement-fixing antigen for typing isolates, the necessity of blind passing many viruses in order to establish mouse pathogenicity was eliminated.