AdUTPase gene ( du) deleted ovine lentivirus (OvLV(Deltadu)) mutant, derived from Visna/maedi virus (VMV) molecular clone KV1772, was constructed. Subsequently, a copy of the optimized green fluorescent protein ( egfp) coding region was fused into the viral pol open reading frame (ORF) at the deleted du locus to generate viral mutant, OvLV(Deltadu-egfp). OvLV(Deltadu) reverse transcriptase (RT) activity and titer of infectious virus in goat synovial membrane (GSM) cell cultures were not affected compared to that of KV1772 and OvLV-85/34 strain (p < 0.05). By contrast, OvLV(Deltadu-egfp) RT activity and virus titer were lower than for KV1772 and OvLV(Deltadu) (p < 0.05). OvLV-85/34 RT in sheep monocyte-derived macrophages (SMDM) was higher than that of KV1772, OvLV(Deltadu) and OvLV(Deltadu-egfp) (p < 0.05). The ability to prevent dUTP mis-incorporation into newly synthesized DNA was disrupted in OvLV(Deltadu) and OvLV(Deltadu-egfp) (p < 0.05). Immunoprecipitation demonstrated that GFP is expressed by OvLV(Deltadu-egfp) at a low level. OvLV(Deltadu-egfp) retained egfp after 10 passages in cell culture.OvLV(Deltadu-egfp) was re-isolated in GSM cells from peripheral blood mononuclear (PBMN) cells of three of four OvLV(Deltadu-egfp)-inoculated lambs, but by contrast to the in vitro experiments OvLV(Deltadu-egfp) lost the insert. Priming with OvLV(Deltadu-egfp) did not prevent infection with pathogenic OvLV, but cell-associated viremia in a mock-infected contact control lamb was higher than in OvLV(Deltadu-egfp)-primed lambs. OvLV serum antibody titers increased steadily in OvLV(Deltadu-egfp)-inoculated lambs, but in a lamb from which OvLV(Deltadu-egfp) was not reisolated the antibody titer surpassed the negative/positive cut-off value only after challenge with OvLV-85/34. Because OvLV(Deltadu-egfp) is attenuated for pathogenicity in vitro, replicates in vivo and stimulates an antibody response, subsequent experiments need to address the likelihood of using OvLV(Deltadu-egfp) as an attenuated, live-virus vaccine to protect sheep against OvLV-induced disease when challenged with pathogenic OvLV.