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The growth of Rio Grande (RG) virus, the only phlebovirus known to occur in the United States, was studied in Lutzomyia anthophora, its suspected sand fly vector. RG viral titers in infected flies increased more than 10,000-fold within 7 days after intrathoracic inoculation. Experimentally infected female L. anthophora transmitted virus transovarially (vertically) to 54.8% of their F1 progeny. This is the first virologically confirmed demonstration of transovarial transmission of a phlebovirus by sand flies. It indicates one mechanism by which RG and possibly other phleboviruses may be maintained in nature.