Isolated limb perfusion (ILP) is a limb salvage surgical modality used to deliver chemotherapy and biologic agents to locally advanced and recurrent extremity soft tissue sarcoma (STS), and may be readily tailored for delivery of gene therapy. We set out to test the feasibility of delivering AdFLAGp53 (replication incompetent adenovirus bearing FLAG-tagged wild-type p53) and Ad.hTC.GFP/E1a.RGD (a fiber-modified, replication selective oncolytic adenovirus) into human leiomyosarcoma xenografts by ILP. Nude rats bearing SKLMS-1 tumors in their hind limbs underwent ILP with escalating doses of AdLacZ or AdFLAGp53 (study 1), or with Ad.CMV.GFP.RGD or Ad.hTC.GFP/E1a.RGD (study 2) following in vitro confirmation of therapeutic potential in STS cell lines and strains. Seventy-two hours after delivery, reverse transcription-polymerase chain reaction confirmed FLAGp53 expression, and immunohistochemistry confirmed diffuse upregulation of p21CIP1/WAF1 in ILP-treated tumors. Ad.hTC.GFP/E1a.RGD perfused tumors demonstrated robust macroscopic transgene expression throughout their substance, but not in perfused normal tissues, 21 days after delivery. Intra-tumoral viral replication was confirmed by immunohistochemical staining for early (E1a) and late (hexon) viral protein expression. Terminal deoxynucleotidyl transferase-mediated-digoxigenin nick end-labeling staining identified foci of cell death within regions of viral replication. In conclusion, therapeutic adenoviral gene therapy against limb borne human STS can be successfully delivered by ILP and warrants further investigation.