An enzyme-linked immunosorbent assay (ELISA) was developed using a recombinant protein corresponding to the N'-terminal hydrophilic region of transmembrane glycoprotein (TM) of ovine lentivirus. This assay reproducibly detected antibodies in sera from 207 of 212 ovine progressive pneumonia (OPP) virus-infected sheep, and the recombinant TM ELISA accurately identified 26% (35 vs. 9) more seropositive samples than did the agar gel immunodiffusion test when applied to 100 sera from an infected flock. This assay also yielded no false-positive results in 14 true negative sera. Results of these experiments were further confirmed by the recombinant TM and recombinant p25 Western blot assay. A single recombinant TM antigen, as the coating antigen in ELISA, can be used successfully for the detection of OPP virus-infected animals and can improve the sensitivity and specificity for OPP diagnosis.