ABSTRACT This study aimed to isolate cells from the Wharton's jelly of umbilical cord (WJUC) of sheep collected during natural parturition using different culture media, in addition to reporting for the first time the permissiveness of these cells to in vitro infection by small ruminant lentiviruses. Ten umbilical cords were collected from healthy sheep. Each cord explants were grown in different media consisting of MEM, low glucose DMEM, M199, and RPMI-1640. The permissiveness of infection of sheep cells from WJUC was tested with CAEV-Cork and MVV-K1514 strains, inoculating 0.1 MOI of each viral strain. Four supernatants from each strain were obtained from WJUC sheep cell cultures infected in different media. The results demonstrated the presence of cytopathic effect after the in vitro infection by CAEV-Cork and MVV-K1514 with all of the tested culture media. Nested-PCR detected proviral DNA in all supernatants. Supernatants containing CAEV-Cork viruses had TCID50/ml titres of 105.5 in MEM, 104.0 in low glucose DMEM, 105.0 in M199, and 105.7 in RPMI-1640. Supernatants containing the MVV-K1514 virus had TCID50/ml titres of 104.3 in MEM, 103.5 in low-glucose DMEM, 104.7 in M199, and 103.5 in RPMI-1640. Sheep cells from WJUC are permissive to in vitro infection by small ruminant lentivirus.