Adenoviral vectors can efficiently infect myeloma cell lines, but transduction of fresh myeloma cells performed at low multiplicity of infections (MOIs) showed only partial efficacy. The modified adenoviral vector AdZ.F(pK7), through binding of polylysines to heparan sulfate-containing receptors, could increase virus adsorption and gene transfer efficiency in myeloma cells, which express heparan sulfate-containing receptors. Thus, we investigated the ability of AdZ.F(pK7) vector to achieve efficient gene transfer in primary cultured fresh myeloma cells. Transduction of 16 primary cultured myeloma samples showed that gene transfer was much more efficient with AdZ.F(pK7) than with control AdZ.F. Both addition of soluble heparin and cell treatment with heparinase I dramatically inhibited gene transfer in myeloma cells by AdZ.F(pK7) but had no effect with AdZ.F, while addition of recombinant fiber protein inhibited AdZ.F but not AdZ.F(pK7), confirming that AdZ.F(pK7) gene transfer in myeloma cells is mediated by the targeting of heparan sulfates. AdZ.F(pK7) transduction of bone marrow cells showed that myeloma cells and hematopoietic progenitor AC133-, CD34-, and CD33-positive cells were efficiently transduced at an MOI of 100, but that only myeloma cells were significantly transduced at an MOI of 12. Thus, AdZ.F(pK7) vector seems to be well suited for immunological approaches of gene therapy or bone marrow-purging applications in multiple myeloma.