Two species of Rhinoestrus (i.e. Rhinoestrus purpureus (Brauer) and Rhinoestrus usbekistanicus Gan) cause nasal myiasis in horses, donkeys and zebras. In the past 15 years myiasis caused by R. purpureus has been reported in Egypt and by R. usbekistanicus in Senegal and Niger, both in horses and in donkeys. With the aim to investigate the presence of this myiasis in autochthonous horses and donkeys from southern Italy and to study the seasonal trend of larval infection, 212 native horses were necropsied in two slaughterhouses in the Apulia region (site A) from January to November 2003, and 120 native horses and two donkeys in one slaughterhouse in Sicily (site B) from January to October 2003. Thirteen of 212 and 5 of 120 horses examined from sites A and B, respectively, were infected by nasal bot fly larvae. Both donkeys examined were positive. Two hundred and thirteen larvae, representing all stages, were collected from the throat region, the turbinates and beyond the lamina cribrosa of the ethmoid bone, in the cerebral cavity. Third larval stages were retrieved from April to September with the highest mean burden in site A in May and in July in site B. The simultaneous presence all three larval stages at site B suggests the existence of two or more overlapping generations. Larvae were identified on the basis of peritreme structures and arrangement of the spines on the dorsal surface of the third segment. Some L3 collected from sites A and B presented morphological characteristics of both R. usbekistanicus and R. purpureus. The presence of mixed features on the L3 of Rhinoestrus collected from the same animals may be explained by the existence of a single species of Rhinoestrus presenting different morphotypes or of two species of Rhinoestrus which are both present in southern Italy since this area is midway between eastern and African countries where these two species have been reported.