PURPOSE: Interleukin-12 (IL-12) promotes adaptive type-1 immunity and has demonstrated anti-tumor efficacy, but systemic administration leads to severe adverse events (AEs), including death. This pilot trial investigated safety, efficacy, and immunologic activity of intratumoral delivery of IL-12 plasmid DNA (tavo) via in vivo electroporation (i.t.-tavo-EP) in patients with Merkel cell carcinoma (MCC), an aggressive virus-associated skin cancer. EXPERIMENTAL DESIGN: Fifteen MCC patients with superficial injectable tumor(s) received i.t.-tavo-EP on days 1, 5, and 8 of each cycle. Patients with locoregional MCC (Cohort A, N=3) received one cycle before definitive surgery in week 4. Patients with metastatic MCC (Cohort B, N=12) received up to 4 cycles total, administered at least six weeks apart. Serial tumor and blood samples were collected. RESULTS: All patients successfully completed at least one cycle with transient, mild (Grade 1, 2) AEs and without significant systemic toxicity. Sustained (day 22) intratumoral expression of IL-12 protein was observed along with local inflammation and increased tumor-specific CD8+ T cell infiltration, which led to systemic immunologic and clinical responses. The overall response rate was 25% (3/12) in Cohort B, with two patients experiencing durable clinical benefit (16 and 55+ months respectively). Two Cohort A patients (one with pathologic complete remission) were recurrence-free at 44+ and 75+ months. CONCLUSIONS: I.t.-tavo-EP was safe and feasible without systemic toxicity. Sustained local expression of IL-12 protein and local inflammation led to systemic immune responses and clinically meaningful benefit in some patients. Gene electrotransfer, specifically i.t.-tavo-EP, warrants further investigation for immunotherapy of cancer.