Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human alpha1-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 x 10(12) particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity.