Adenoviral vector-mediated gene delivery is currently the focus of many efforts to administer therapeutic gene products for the treatment of cancer. Although these vectors are replication deficient, they can induce specific immune responses against both vector- and transgene-encoded proteins. We have extended these findings to determine the level of innate natural killer (NK) cell responses to adenoviral vector administration in vivo. Similar to many replicating viruses, the vectors induce prominent NK cell activation in mouse spleens within 2 days of injection. We also observed these NK cell responses regardless of the route of administration. Furthermore, stimulation of NK cells by adenoviral vectors is independent of viral gene transcription, as UV inactivation of the vectors does not reduce the NK cell response. In contrast, heat treatment of the vectors destroys their ability to activate NK cells, demonstrating the necessity for intact vector particles. In addition, we found that administration of "empty" (no transgene) adenoviral vectors delays tumor growth in mice bearing B16 melanomas, and this effect is abrogated by depletion of NK cells. Collectively, these results demonstrate in a murine system that the adenoviral vector gene delivery system itself stimulates NK cells, and this in turn can nonspecifically enhance antitumor immunity.