To investigate the respective role of transduced cells in the induction of immune response following intramuscular inoculation of adenovirus-based vaccines, we generated several replication-defective adenoviruses expressing the glycoprotein D gene of pseudorabies virus under the control of four different promoters: major late promoter of adenovirus type 2, human cytomegalovirus immediate-early promoter/enhancer (CMV), Rous sarcoma virus-long terminal repeat promoter, and human desmin gene 5' regulatory region (DES). All the adenovirus constructs were able to fully protect mice, in the contrary of direct DNA inoculation of plasmids harboring the same transcription units. The far most effective adenovirus constructs, on the criterion of protective doses and specific antibody response induction, were those in which the foreign gene was driven by the DES or CMV promoter. Wide variations in promoter strength in vitro were evidenced in several cell culture types representative of putative target cells following muscular inoculation (myoblasts, myotubes, fibroblasts, macrophages, and endothelial cells). The level of efficacy in vivo, was not correlated with the level of expression in vitro in myotubes, but paralleled the level of expression in endothelial cells and in myoblasts. Together with previously published data, these results suggest that, following adenovirus injection, locally produced cytokines may induce myoblasts to act as local antigen presenting cells.