The Epstein-Barr-transformed B lymphoblastoid cell lines, LCL, which express antigens, are potential antigen-presenting cells (APCs) for the induction of cytotoxic T lymphocytes in vitro. However, transfecting LCL with subsequent selection by antibiotics is notoriously difficult because the plating efficiencies of LCL are reported to be 1% or less. Therefore, this study investigated the optimal conditions for increasing the transduction efficiency of a recombinant adenovirus to LCL for use as a source of APCs. The transduction efficiencies were < 13% (SD $pm$ 2.13) at a multiplicity of infection (MOI) of 100, while it was increased to 28% (SD $pm$ 9.43) at an MOI of 1000. Moreover, its efficiencies to LCL that expressed the coxsackie adenovirus receptor were increased to 60% (SD $pm$ 6.35) at an MOI of 1000, and were further increased to 70% (SD $pm$ 4.56) when combined with the centrifugal method. The cationic liposome or anionic polymer had no effect on the transduction efficiency when compared to that of the centrifugal method. These results may be used as a convenient source of target cells for a CTL assay and/or autologous APCs for the induction of the in vitro CTL responses that are specific to viral and tumor antigens.