Maedi-visna is a systemic disease of sheep caused by a lentivirus, maedi-visna virus (MVV), which mainly affects the lungs and central nervous system but may also affect the mammary glands, joints and other tissues. The aim of the present study was to determine whether the third eyelid was affected in cases of systemic infection. Third eyelid and lung samples from sheep naturally infected with maedi were used. Total DNA was extracted from paraffin-wax-embedded tissues, and a nested polymerase chain reaction (PCR) was performed to amplify MVV proviral DNA. The samples were also tested by in-situ PCR and immunohistochemical methods specific for the detection of MVV proviral DNA and p25, respectively. All sheep showed moderate to severe chronic lymphoproliferative inflammation in the third eyelids. Products of the expected size were obtained by PCR from both lung and third eyelid tissue. In the nictitating membrane, MVV proviral DNA was detected in situ within macrophages, and glandular, ductal and surface epithelia. Immunohistochemistry demonstrated that the infection was productive. Taken together, these results indicate that the third eyelid may represent a target for natural MVV infection and may play a role in disease transmission.