The development of novel treatment strategies for the effective delivery of new therapies directed against solid tumors, particularly metastatic melanoma, are required. A novel therapeutic property has previously been discovered for the HIV-1 accessory protein viral protein R (Vpr), based on the ability of this protein to induce G(2) cell cycle arrest as well as apoptosis in various tumor cell lines. Likewise, in vivo electroporation has been utilized as an effective delivery platform for DNA plasmids expressing potentially therapeutic proteins and has been targeted to normal tissues as well as tumors. Our previous findings demonstrated that delivery of a Vpr expression plasmid (pVpr) to established subcutaneous B16.F10 melanoma tumors by in vivo electroporation yielded long-term complete tumor regression in a small percentage of mice. In this study, we modified the electroporation regimen for pVpr with the goal of enhancing the anti-tumor activity of Vpr. pVpr was injected intratumorally, on days 0, 2 and 4, into established subcutaneous B16.F10 melanoma tumors followed by in vivo electroporation. Treatment with 100 mg of pVpr plus electroporation on the modified treatment days resulted in 50% of the mice undergoing complete tumor regressions coupled with long-term survival (i.e., greater than 100 days post treatment). Additional investigations established the intratumoral expression of Vpr and induction of apoptosis for a period of at least seven days after the modified pVpr treatment regimen. This report demonstrates that the anti-tumor activity of a pVpr plus electroporation regimen against established subcutaneous B16.F10 melanoma tumors can be significantly enhanced by a modified treatment schedule. In addition, it appeared that this enhanced anti-tumor effect was correlated with prolonged Vpr expression and the associated induction of intratumoral apoptosis.