The catalytic component of human telomerase reverse transcriptase (hTERT) is not expressed in most primary somatic human cells, whereas the majority of cancer cells reactivate telomerase by transcriptional up-regulation of hTERT. Several studies demonstrated that the hTERT promoter can be used to restrict gene expression of E1-deleted replication defective adenoviral vectors to telomerase-positive cancer cells. In this study, a conditionally replicating adenovirus (hTERT-Ad) expressing E1A genes under control of a 255-bp hTERT-promoter was constructed. Additionally, an internal ribosomal entry site-enhanced green fluorescent protein cassette was inserted downstream of the E1B locus to monitor viral replication in vivo. Adenoviral replication of hTERT-Ad and enhancement of enhanced green fluorescent protein expression could be observed in all investigated telomerase-positive tumor cell lines. In contrast, hTERT-Ad infection of telomerase-negative primary human hepatocytes did not result in significant replication. The capability of hTERT-Ad to induce cytopathic effects in tumor cells was comparable with that of adenovirus wild type and significantly higher compared with ONYX-015, regardless of the p53 status of the tumor cells. Single application of low-dose hTERT-Ad to tumor xenografts led to significant inhibition of tumor growth, confirming the potential therapeutic value of conditionally replicative adenoviral vectors. These in vivo experiments also revealed that hTERT-Ad-mediated oncolysis was more efficient than ONYX-015 treatment. These results demonstrate that expression of E1A under transcriptional control of the hTERT promoter is sufficient for effective telomerase-dependent adenovirus replication as a promising perspective for the treatment of the majority of epithelial tumors.