Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 105PFU of virus, an E1A null mutant,pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant,pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU,pmE109 andpmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.