Toscana virus was maintained in a laboratory colony of Phlebotomus perniciosus by vertical (transovarial) transmission for 13 consecutive generations over a 23-month period. No significant biological changes were noted in the virus after prolonged vertical passage in the sand flies, and transovarially infected females were able to transmit the agent by bite to susceptible animals. Chronic infection of Ph. perniciosus with Toscana virus had no apparent effect on the insects' rate of eclosion. In the absence of selection and with random matings, the virus infection rates in each subsequent generation of the colony decreased, suggesting that Toscana virus cannot be maintained in Ph. perniciosus by transovarial transmission alone. Alternative mechanisms for virus maintenance are discussed.