A heterologous competition radioimmunoassay (RIA) which consisted of 125I-labeled langur retrovirus major gag protein and goat anti-squirrel monkey retrovirus serum was used to detect a type D retrovirus-associated antigen in tumor cell homogenates, lung fluid, and cell culture supernatant fluids of naturally occurring and experimentally-induced ovine pulmonary carcinoma (OPC, sheep pulmonary adenomatosis). In this assay, there was no cross reactivity between structural proteins of the type D retrovirus and an ovine lentivirus, which frequently co-infects OPC-affected sheep. The sensitivity of the assay was similar to an immunoblotting assay using antiserum to Mason-Pfizer monkey virus major gag protein which had been used previously to detect the OPC retrovirus antigen in tumor homogenates and lung fluids of OPC-affected sheep. All unconcentrated samples of lung fluid collected from five sheep with naturally occurring OPC or six sheep with experimentally induced OPC competed in the competition RIA. The competition RIA titers of the type D retrovirus antigen in lung fluids of lambs with induced OPC were relatively higher than the titers of this antigen in the naturally occurring OPC cases. The competition RIA detected the retrovirus antigen associated with OPC in the culture fluids of four out of five primary lung cultures from OPC sheep tested between 1 and 56 days after culture initiation. Because this RIA is appropriate for the quantitation of OPC-associated antigen, it will provide a means for determination of the target cell type for OPC virus replication in vitro.