Murine lymphocytes are relatively refractory to efficient transfection or retroviral gene transduction. Adenovirus has been used as a vector to transduce a wide variety of cell types. Several advantages of adenoviruses are their ability to transduce non-cycling cells and to transduce the majority of cells in a population. Unfortunately, lymphocytes are not susceptible to infection with conventional adenovirus. Therefore, to express genes efficiently in murine B cells, we tested the ability of genetically modified adenovirus to transduce the beta-galactosidase gene. We found that adenovirus containing polylysine in the fiber knob was able to efficiently transduce lipopolysaccharide (LPS)-activated splenic B cells and the B lymphoma line M12.4.1; greater than 80% of the cells expressed beta-galactosidase activity. However, small resting B cells did not express activity unless treated with LPS after infection. This transduction was mediated by interaction with charged molecules since heparan-sulfate, and to a lesser degree chondroitan sulfate, inhibited the transduction. In addition, adenovirus containing a FLAG epitope in the fiber protein was used to target the FcR expressed on B cells using an anti-FLAG antibody. In the presence of anti-FLAG, the modified adenovirus was able to efficiently transduce LPS-activated B cells and several B cell lymphoma lines. Interestingly, in the absence of anti-FLAG, there was low level transduction in the LPS-blasts and in M12.4.1 that was not inhibited by soluble adenovirus fiber protein or agents that block RGD-integrin interactions. These results demonstrate that modified adenovirus efficiently transduce B lymphocytes which will be critical for targeting genes to normal or malignant B cells.