Recombinant adeno-associated virus (rAAV) vectors have gained an extensive record of safety and efficacy in animal models of human disease. Infrequent reports of genotoxicity have been limited to specific vectors associated with excess hepatocellular carcinomas (HCC) in mice. In order to understand potential mechanisms of genotoxicity, and identify patterns of insertion that could promote tumor formation, we compared a self-complementary AAV (scAAV) vector designed to promote insertional activation (scAAV-CBA-null) to a conventional scAAV-CMV-GFP vector. HCC-prone C3H/HeJ mice and severe combined immunodeficiency (SCID) mice were infected with vector plus secondary treatments including partial hepatectomy (HPX) and camptothecin (CPT) to determine the effects of cell cycling and DNA damage on tumor incidence. Infection with either vector led to a significant increase in HCC incidence in male C3H/HeJ mice. Partial HPX after infection reduced HCC incidence in the cytomegalovirus-green fluorescent protein (CMV-GFP)–infected mice, but not in the cognate chicken β-actin (CBA)-null infected group. Tumors from CBA-null infected, hepatectomized mice were more likely to contain significant levels of vector DNA than tumors from the corresponding CMV-GFP–infected group. Most CBA-null vector insertions recovered from tumors were associated with known proto-oncogenes or tumor suppressors. Specific patterns of insertion suggested read-through transcription, enhancer effects, and disruption of tumor suppressors as likely mechanisms for genotoxicity.