Deoxyribonucleic acid (DNA) vaccination offers the prospect of novel, safe vaccines that can stimulate cell-mediated immunity as well as antibody. An understanding of how and where transfection occurs in vivo will assist strategies to enhance their efficacy. Plasmid DNA encoding for the expression of firefly luciferase was injected intramuscularly into sheep. The DNA was rapidly disseminated and could be detected in efferent lymph within 5 min. At the earliest time that cells were tested separately from lymph plasma (1 h), DNA was found to be present in both the lymph plasma and within lymph cells. Although plasmid DNA could be detected intracellularly in lymph and in muscle tissue at the site of inoculation, no luciferase gene expression could be detected. The results suggest that, in addition to the internalization of plasmid DNA, other factors may limit gene expression, possibly the rate of DNA migration into the nucleus.