Cutaneous leishmaniasis (CL) is endemic to the Yucatan Peninsula of Mexico. The main causative agent is the parasite Leishmania mexicana (Biagi) (Kinetoplastida: Trypanosomatidae) and, based on the classic work of Dr Biagi's research team, it has been generally accepted and frequently reported that the only vector of L. mexicana in the region is the sandfly Lutzomyia olmeca olmeca (Vargas & Diáz-Nájera) (Diptera: Psychodidae). Evidence gathered from recent entomological studies conducted mainly in Calakmul, Campeche, however, suggests that other species may also be vectors of L. mexicana. We conducted a field study in two villages in Calakmul, Campeche in the Yucatan Peninsula, where recent cases of CL have been reported, to document the species composition and relative abundances of the sandfly fauna and to identify which species are likely to be the main vectors by assessing the biting rates and parasite infection rates of the suspected vector species. Sandfly catches were conducted from November 2005 to February 2006 in Unión 20 de Junio and Dos Lagunas Sur. Sandflies were captured using Shannon (18.00-22.00 hours), Disney and CDC light traps (18.00-06.00 hours). Biting and infection rates were calculated for the four most abundant species: Lutzomyia cruciata (Coquillett), Lu. o. olmeca, Lutzomyia panamensis (Shannon) and Lutzomyia shannoni (Dyar). In Dos Lagunas Sur, Lu. panamensis and Lu. o. olmeca exhibited the highest biting rates throughout the sampling period. In Unión 20 de Junio, Lu. cruciata and Lu. o. olmeca had the highest biting rates over the same period. Regarding infection rates, we report herein the establishment of a polymerase chain reaction protocol and validation of IR1 and LM17 oligonucleotides to analyse the infection rates of sandflies. Out of 769 females analysed, the overall infection rates were 1.4% in Dos Lagunas Sur and 5.3% in Unión 20 de Junio. In Dos Lagunas Sur we found L. mexicana infections in two sandfly species, Lu. shannoni and Lutzomyia ylephiletor (Fairchild & Hertig), whereas in Unión 20 de Junio we found infections in Lu. shannoni, Lu. cruciata, Lu. o. olmeca and Lu. panamensis. The possible role of these four sandfly species in relation to L. mexicana transmission in Calakmul is discussed.