The Iberian lynx (Lynx pardinus) is the most endangered felid species in the world. Lynx populations have decreased dramatically in size and distribution in the last four decades, thus becoming increasingly vulnerable to catastrophic events such as epizooties. From 1989 to 2000, serum samples were obtained from 48 free-ranging lynx captured in the Doñana National Park (DNP, n = 31) and mountains of Sierra Morena (SM, n = 17) in southern Spain. Samples were tested for antibodies against Toxoplasma gondii, feline herpesvirus 1 (FHV-1), feline calicivirus (FCV), feline/canine parvovirus (FPV/CPV), feline coronavirus, feline immunodeficiency virus (FIV), feline leukaemia virus and canine distemper virus (CDV) and for FeLV p27 antigen, to document baseline exposure levels. Antibodies against T. gondii were detected in 44% of lynx, with a significantly greater prevalence in DNP (61%) than in SM (12%). In DNP, prevalence was significantly higher in adult (81%) than in juvenile and sub-adult (41%) lynx, but no such difference was observed in SM. Low prevalences (≤11%) of minimally positive titres were found for FHV-1, FCV and FPV/CPV. This, combined with the lack of evidence for exposure to CDV, FIV and FeLV, suggests that these lynx populations are naïve and might be vulnerable to a disease outbreak in the future. Because of the reduced size of lynx populations, the documented low level of genetic variation (particularly in the DNP population) coupled with the recently documented state of immune depletion in a majority of necropsied lynx, it is important to better understand the threat and potential impact that disease agents might pose for the conservation of this endangered species. Future surveillance programs must include possible disease reservoir hosts such as domestic cats and dogs and other wild carnivores.