Canine leishmaniasis (CanL) caused by Leishmania infantum is a serious zoonotic public health and veterinary problem in the Mediterranean basin. Leishmania infection in domestic cats (Felis catus domesticus) has been reported in several countries where this zoonosis is endemic, such as Portugal, Spain, Italy, France, Greece, Israel, Palestine and Brazil. The aim of this study was to contribute to the knowledge of the role played by cats in Leishmania epidemiology, in an endemic focus of zoonotic leishmaniasis, the Lisbon metropolitan area, Portugal. L. infantum DNA was detected in peripheral blood of 28 out of 138 cats (20.3%). The result of PCR in blood of cats was not closely associated with the level of specific circulating antibodies in their sera. Positive serology was observed only in one cat out of 76. In the same geographic region and time period the indirect immunofluorescent test revealed 20.4% (31/152) of dogs with antibodies and PCR detected Leismania DNA on 34.9% (53/152) animals. Despite the fact that specific antibodies have been validated for diagnosis of CanL, their detection does not seem to be sensitive enough to predict Leishmania infection in cats. On the other hand, the presence of parasite DNA in cat's peripheral blood during the transmission season and out of the season suggests that these animals living in endemic areas are frequently exposed or infected with the parasite. Although dogs have been universally regarded as the major domestic/peridomestic reservoir hosts, the present data allow us to hypothesize that cats can act as an alternative reservoir host of L. infantum, rather than an accidental host. However, in order to evaluate the existence of a transmission cycle with cats sustaining and spreading zoonotic leishmaniasis is necessary to prove that these animals can transmit the parasite to the vector in nature.