Infection by different Leishmania spp. in cats has been reported in many countries. In Spain, since the first Leishmania infection described in 1933, sporadic clinical cases in cats have been reported. Various serologic studies performed in other areas of Spain have shown seroprevalences ranging between 1.7 and 60%. The aim of the present study was to determine the prevalence of leishmaniasis in cats from Central Spain (Madrid), and to assess the existence of associations between Leishmania infantum infection and relevant data obtained from each cat. Two-hundred thirty-three cats attended at the Veterinary Teaching Hospital in Madrid between September 2005 and June 2006 were tested for L. infantum using the indirect immunofluorescent antibody (IFA) test (cutoff: 1:100) and PCR. PCR testing was performed on the samples to detect Leishmania infection, targeting the kinetoplast DNA (kDNA). Our results showed a seroprevalence of 1.29% (3/233) using IFA test. Another seven cats were also seroreactive to L. infantum one dilution under the cutoff (1:50). Considering all the seroreactive samples, the percentage of positive animals to L. infantum was 4.29%. Only one of the cats (0.43%) included in the study was PCR-positive. Relative lymphocytosis and an increase in alanine aminotransferase (ALT) value were statistically associated with seroreactivity to L. infantum. Our results demonstrate the presence of cats seroreactive to L. infantum in Central Spain, an endemic area for this disease in dogs.