Infection by Leishmania species is increasing worldwide. It was hypothesized recently that cats act as a secondary reservoir for Leishmania infection. The aim of the present study was to assess the prevalence of Leishmania infantum antibodies and DNA in blood samples collected in a sample of stray cats in metropolitan area of Milan in northern Italy, which is a nonendemic area for leishmaniasis. An indirect immunofluorescence antibody test for L. infantum showed that 59 of 233 cats (25.3%) were seroreactive, 38 samples (16.3%) had antibody titers of 1 : 40, 15 (6.4%) had antibody titers of 1 : 80, and 6 (2.6%) had antibody titers of 1 : 160. Feline immunodeficiency virus (FIV) seropositive status was statistically associated with seroreactivity to L. infantum (P = 0.01) as shown by univariate and multivariate logistic regression (P = 0.0098; OR = 7.34). All blood samples that were tested using real-time PCR were negative for parasite DNA. These results were surprising, since no autochthonous human or canine cases of leishmaniasis have ever been reported in this region of northern Italy. It is possible that this high seroreactivity to L. infantum could be due to cross-reaction with antigens from other parasites. Additional studies that include parasite isolation are needed to clarify our findings on feline leishmaniasis in this region.