Five isolations of the Alagoas serotype of vesicular stomatitis virus (Rhabdoviridae: Vesiculovirus) were made from naturally infected phlebotomine sand flies (Lutzomyia spp.) collected in Colombia. These are the first isolations of Alagoas virus from an arthropod. Replication of the virus occurred in laboratory-reared sand flies (Lutzomyia longipalpis) after inoculation. Bite and transovarial transmission of the virus was also demonstrated in experimentally infected sand flies. Alagoas virus neutralizing antibodies were found in sera of humans and animals living near the insect collection site; antibody rates among human residents of two nearby towns were 63% and 83%, respectively. Results of comparative serologic studies demonstrated that Alagoas virus is closely related antigenically to Indiana, Cocal, and Maraba viruses and that these four agents form a complex within the vesicular stomatitis virus serogroup. The antigenic similarity among these four viruses makes their differentiation difficult; it also raises doubts about the accuracy of current laboratory methods used for identifying isolates in this serogroup. A discussion follows on the significance of human antibodies to these agents and on the role of sand flies in their ecology.