The route of tomato spotted wilt virus (TSWV) in the body of its vectors, Frankliniella occidentalis and Thrips tabaci (Thysanoptera: Thripidae) was studied during their development. First instar larvae were allowed, immediately upon hatching, to acquire virus from mechanically infected Datura stramonium plants for 24 h. The rate of transmission by adults was determined in inoculation access feeding test on Emilia sonchifolia leaf disks. Thrips tissues were analysed for infection at 24 h intervals after the acquisition-access feeding period, and assayed by the whole-mount immuno-fluorescent staining technique. The virus was initially detected in the proximal midgut region in larvae of both species, and then in the second and third midgut regions, foregut, and salivary glands. Occasionally the first infections of the salivary glands were already detected in one-day-old second stage larvae. The intensity of the infection in the various organs of the thrips of each species was positively related to the transmission efficiency. In both thrips populations good agreement was found between the percentage of second instar larvae and adults with at least one infected salivary gland lobe and the percentage of transmitting adults. These results support the contention that the virus must reach the salivary glands before thrips pupation in order to be transmitted by old second instar larvae and adults.