To correlate the presence of ovine lentivirus (OvLV) as detected by polymerase chain reaction (PCR) with detection of antibody, 42 sheep from a flock with enzootic OvLV infection were studied. The results of agar gel immunodiffusion (AGID), ELISA, and immunoblotting assays were compared, and leukocytes (blood, bone marrow, lymph node, and lung cells) were assessed for viral DNA by PCR using pol and LTR primers; amplified products were detected by specific DNA and RNA probes. Based on the number of animals that had detectable viral DNA, the specificities of AGID, ELISA, and immunoblotting were 77%, 92%, and 95 or 100% (depending on which criterion was used to interpret immunoblot results), respectively. Only in animals with OvLV-associated disease was OvLV DNA detected in leukocyte DNA prior to the amplification of virus in culture and only in this group was high titer antibody detected to the OvLV major surface (gp 105) and transmembrane (gp 55) antigens. Animals that were both antibody and PCR-negative lacked histopathologic evidence of disease. From this study there was no indication that OvLV infection without the development of antibody occurs, and detection of OvLV DNA in animals with weak or partial serological reactions likely indicates early OvLV infection rather than false-positive PCR results.