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Thelazia callipaeda Railliet and Henry (Spirurida: Thelaziidae), commonly called oriental eyeworm for its widespread presence in the Far East, has been recently found to affect dogs, cats and foxes in northern and southern Italy. Although the biology of T. callipaeda in the definitive hosts has been recently investigated, many doubts still remain about its biology in insect vectors. It has been suggested that more than one species of Diptera, namely Musca domestica Linnaeus (Diptera: Muscidae) and Amiota okadai Maca (Diptera: Drosophilidae), may be involved in the transmission of T. callipaeda in China. The aim of the work described here was to verify the role of M. domestica as a vector of T. callipaeda both in experimental and natural conditions. A total of 310 M. domestica (Group 1) were put in a cage and allowed to feed for 14 days around the eyes of a dog naturally infected by T. callipaeda. Ten flies were collected daily for 14 days. A total of 149 houseflies (Group 2) were fed with T. callipaeda first stage larvae (L1) and dissected at 1, 2 and 7 days post-infection. From June to August 2003, flies were netted (Group 3) in two different sites every 10 days both from the environment and directly from the periocular region of dogs affected by thelaziosis. Musca flies were examined for eyeworms by dissection and visual inspection of house flies (Groups 1 and 2) and using a molecular approach (Groups 1–3) via a specific amplification of the ribosomal internal transcribed spacer 1 (ITS1) sequence of T. callipaeda. On the whole, 180 pools of M. domestica flies were processed molecularly and all the experimentally infected flies (Groups 1 and 2) were found to be negative both at the visual dissection and at the molecular assay. Similarly, the 234 M. domestica collected from Group 3 were negative for T. callipaeda. The results clearly suggest that M. domestica is unlikely to act as a vector of T. callipaeda in southern Europe, in contrast with a single previous report.