The relative paucity of receptors for the commonly used adenovirus serotype 5 in many cancer types undermines the efficacy of Ad5-based approach for cancer gene therapy. We have previously shown that coxsackie-adenovirus receptor (CAR) is expressed at decreased levels in several primary head and neck squamous cell carcinoma (HNSCC) cell lines established from tumor samples and that retargeting adenoviral infection to the CD46 receptor using the Ad5/35 hybrid virus results in highly efficient transfection of these cells. We sought to examine the effect of this retargeting in the context of the conditionally replicating adenovirus (CRAD) approach. By subjecting the viral E1A gene under the regulation of human telomerase reverse transcriptase promoter we produced Ad5/35-TERT, a transductionally targeted CRAD. The anti-tumor efficacy of this virus was tested in two primary HNSCC cell lines, chosen to represent high (55.2%) and low (3.2%) CAR expression levels. In vitro experiments demonstrated that Ad5/35-TERT is significantly more effective in killing primary HNSCC cells than the non-targeted Ad5-TERT. The difference between the two viruses was clearly more pronounced in HNSCC cells with low CAR expression. In an in vivo experiment using a subcutaneous HNSCC mouse model Ad5/35-TERT was more effective than Ad5-TERT in both high- and low-CAR HNSCC cells. These results demonstrate that enhanced transfection by hybrid virus strategy results in an increased anti-tumor efficacy when using CRADs. The effect is especially distinctive in target cells with low CAR expression.