Leishmaniasis has increased in importance in recent years because infection with human immunodeficiency virus (HIV) has emerged as a risk factor for this disease. However, the actual prevalence of leishmaniasis in the general population of Spain is unknown. We present a study of the seroprevalence of infection with Leishmania infantum in the general population of Castilla-Leon, Spain. A random sample of individuals presenting to health care clinics (4,825 sera) and of HIV-infected patients in the autonomous community of Castilla-Leon was collected in 1996. The sero-prevalence of antibodies to L. infantum was determined by an indirect enzyme immunoassay and found to be 4.9% in the general population. There was a significant increase in seroprevalence with age (P = 0.001), from 3.96% in those 14-20 years old to 7.2% in those > 70 years old. There were no significant differences between women and men (5.0% versus 4.9%; P = 0.9534). Seroprevalence was significantly higher in people from rural areas than in those from cities (6.0% versus 3.4%; P = 0.001). Patients infected with HIV had a seroprevalence for L. infantum of 64.0%. No differences were observed between women and men, and prevalence did not increase with age.