Culicoides variipennis (Diptera: Ceratopogonidae) the primary vector of bluetongue virus (BTV) in the U.S.A. were asynchronously mixedly infected with two BTV serotypes (BTV-10 and BTV-17); flies first ingested a blood meal that contained BTV-17 and 1, 3, 5, 7, and 9 days later selected flies ingested a second blood meal that contained BTV-10. Control flies ingested each parental virus separately, or both viruses simultaneously, in a single blood meal. Electrophoretic analysis of progeny virus clones indicated that superinfection with BTV-10 occurred when the flies ingested the second virus 1, 3 and 5 days post-initial infection. Parental BTV-17 and reassortant virus clones were isolated from these flies, but parental BTV-10 virus was not isolated from any flies. Reassortant clone frequencies were 67%, 71% and 17% when superinfection occurred on days 1, 3 and 5 after initial infection, respectively, as compared to 48% for simultaneously infected flies. Only parental BTV-17 clones were isolated from flies that ingested the second virus on days 7 and 9 after initial BTV-17 infection. The results indicated that interference to superinfection occurred in C. variipennis by 5 days and flies were refractory to superinfection by 7 days post-initial infection. Analysis of segregation of the parental origin of genome segments in the reassortant clones indicated selection against most segments of BTV-10 parental origin. This occurred both in individual flies and in individual groups. The fact that C. variipennis readily fed on a second blood meal and their ability to produce new viral genotypes suggested that these vectors are highly permissive hosts for evolution of BTV by genome reassortment.