We measured the extent of ectromelia virus (ECTV) persistence in experimentally infected BALB/c [H-2^d] by means of in situ PCR and report that ECTV persists in the spleen for at least 12 weeks. Importantly, ECTV maintained a low profile replication in the spleen of mice allowing it to persist considerably longer following initial infection in the absence of clinical disease. Recovery of the virus by cell culture was possible up to 8 weeks post infection. Since ECTV is an Orthopoxvirus that infects genetically susceptible mouse strains, the implication of our findings is that careful screening for this natural mouse pathogen using sensitive assays such as in situ PCR should be periodically carried out in colonies of susceptible mice. On the other hand ECTV and BALB/c [H-2^d] form a suitable study model for generalized viral infection. As such in the light of the data described here the model would suit investigations on the immune status of persistently infected animals.