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Objective: Promising vector vaccine candidates derive from parapoxvirus ovis (PPVO) strain D1701. Thorough analysis of their safety is therefore of great relevance. We investigated the safety of and the immune response induced by the reporter gene β-galactosidase-expressing vector PPVO D1701-VrV. It is important to define the life span of the vector in vaccinated hosts. We chose non-permissive mice for a first study of PPVO longevity and clearence. Methods: Mice were inoculated i.m. with a TCID50 of 106.5 PPVO D1701-VrV and with PBS into the quadriceps and contralateral quadriceps muscle, respectively. The muscles were analyzed for viral genome, infective virus particles and infiltrating leukocytes. Day 21 was fixed as a late vector virus clearance option. The immune cell content of the draining lymph nodes, IFN-γ production by in vitro restimulated splenocytes, and expression of β- galactosidase in myoblasts in vitro was characterized. Results: Upon i.m. application infective PPVO D1701-VrV particles were detectable at the inoculation site for at least seven days but cleared at the fixed late time point. Among the re-isolated vector virus aberrantly shaped PPVO particles were detected, but the reporter gene expression remained stable. The PPVO D1701-VrV-induced local immune activation was characterized by the presence of inflammatory infiltrates and, even more important for the priming capability of the viral vector, significantly enhanced numbers of CD11c+ B220- conventional DC, CD11c+ B220+ plasmacytoid DC, CD4+ T cells, CD8+ T cells, and B220+ B cells in the muscle-draining inguinal lymph nodes. Elevated interferon-gamma (IFN-γ) production by splenocytes of PPVO D1701-VrV-inoculated mice indicated a systemic immunostimulatory effect. The detection of β-galactosidase expression by inoculated myoblasts shows that PPVO D1701-VrV is able to enter murine myoblasts and express the foreign antigen. Conclusions: Our data support the safety of PPVO D1701 as a vector virus under non-permissive conditions.