Ovine herpesvirus-2 (OvHV-2), a member of the gammaherpesviruses (genus Rhadinovirus), asymptomatically infects its natural host, the sheep, but causes malignant catarrhal fever (MCF) in susceptible hosts, such as cattle, deer and pigs. A permissive cell culture system for virus replication has not been identified but viral DNA is present within lymphoblastoid cell lines (LCLs) established from cases of MCF. During this study, a cDNA expression library generated from LCLs was screened with sheep sera and two cDNAs were isolated. One cDNA contained two open reading frames (ORFs) that show similarity to ORFs 58 and 59 of alcelaphine herpesvirus-1 (AlHV-1), a closely related gammaherpesvirus that also causes MCF. Both ORFs 58 and 59 are conserved throughout the gammaherpesviruses. ORF 58 is predicted to be a membrane protein, while ORF 59 has been shown to be an early lytic gene that functions as a DNA polymerase processivity factor. The second cDNA clone contained a partial ORF showing limited similarity to AlHV-1 ORF 73, a homologue of the latency-associated nuclear antigen of human herpesvirus-8, which is associated with latent infections. The full-length OvHV-2 ORF 73 was cloned subsequently by PCR. The ORFs isolated from the library were cloned into a bacterial expression vector and the recombinant proteins tested for their reactivity to sera from OvHV-2-infected animals. An ORF 59 fusion protein was recognized specifically by sera from OvHV-2-infected cattle and will be used to develop a sero-diagnostic test.