The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19 artificial antigen presenting cells was used to numerically expand CD3 T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3 T cells expressed CAR. CAR T cells specifically killed CD19 target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR Vα and TCR Vβ repertoire. Quantitative fluorescence in situ hybridization revealed that CAR T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19 B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).