Polyomaviral vectors are generated by transfecting 293T cells with three sets of DNAs: DNA for the expression of simian virus 40 (SV40) T antigen; DNA for the expression of SV40 capsid proteins, and vector DNA harboring a reporter gene expression cassette carrying a SV40 origin. The vector DNA harbors a minimal sequence originating from SV40, and thus can carry a longer transgene. Moreover, the viable recombinants are not detectable in the vector preparation, and the vectors can transduce the DNA with efficiency similar to that of virions. Vector particles bearing capsid proteins of BK virus, JC virus, and B-lymphotropic papovavirus instead of SV40 were prepared, and they exhibited differential efficiency of gene transduction to the target cells. This method can be used to develop a surrogate system to study the functions of capsid proteins of polyomaviruses and to generate a set of polyomaviral vectors targeted at specific cell types.