We are evaluating the use of electroporation (EP) to deliver a novel DNA vaccine, p.DOM-PSMA(27). This vaccine encodes a domain (DOM) of fragment C of tetanus toxin to induce CD4(+) T cell help, fused to a tumor-derived epitope from prostate-specific membrane antigen (PSMA) for use in HLA-A2(+) patients with recurrent prostate cancer. We report on safety and tolerability and on antibody response to DOM as a first indication of the effect of EP in patients. In this open label phase I/II, two-arm, dose escalation trial DNA was delivered either by intramuscular injection or by intramuscular injection followed by EP (DNA+EP), with five patients per dose level. Three vaccinations were given at 0, 4, and 8 weeks,with booster doses at 24 and 48 weeks; here we allowed crossover between study arms if supported by the safety and immunological data. In the 20 patients in the first two dose cohorts we observed that beyond brief and acceptable pain at the injection site, EP did not appear to add toxicity to the vaccination. We evaluated humoral responses to DOM. Low anti-DOM IgG antibody responses were observed after intramuscular injection of DNA without EP (at week 12: mean 1.7- vs. 24.5-fold increase over baseline with DNA+EP). These could be boosted by delivery of DNA+EP at later time points. Delivery of DNA+EP at all five vaccinations yielded the highest levels of anti-DOM antibody. Responses persisted to 18 months of follow-up. These data establish EP as a potent method for stimulating humoral responses induced by DNA vaccination in humans.