DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional immunoglobulin (Ig) heavy chain gene under the control of a B-cell-specific promoter. Using polymerase chain reaction (PCR), reverse transcriptase-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, Epstein-Barr virus-transformed cell lines, and peripheral blood B lymphocytes of the mature naïve phenotype (IgM(+)/IgD(+)/CD27(-)). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells processed and presented to major histocompatibility complex (MHC)-restricted T lymphocytes a peptide expressed in the transgenic product. This is the first demonstration that primary B lymphocytes possess a program for the spontaneous internalization of DNA, which in turn imparts the cell with new immunological functions. As spontaneous transgenesis is obtained using a nonviral vector, does not require prior cell activation, and is not associated with chromosomal integration, the findings reported here open new possibilities for genetic manipulations of mature naïve B lymphocytes for therapy and vaccination.