We investigated the efficacy of a recombinant adenovirus in inducing a cytolytic T-lymphocyte (CTL) response in mice against tumor antigen PBISA, which is present on mouse mastocytoma P8 15. The recombinant adenoviral vector (Aden0.P I A) contained the sequence coding for the antigenic nonapeptide which binds to the H-Z.Ld molecule to form antigen P815A We verified that murine cells infected in vitro with Adeno.PIA were lysed by an anti-P815A CTL clone. Mice then received a single intradermal injection of Adeno.PIA, and after a few weeks their spleen cells were stimulated in vitro with tumor cells expressing antigen P815A An anti-PBI5A CTL response was observed with the spleen lymphocytes of nearly all the mice, providing the lymphocytes were re-stimulated in vitro with cells expressing both P8 15A and co-stimulatory molecule B7. I. When the stimulatory cells did not express B7. I, a specific CTL response was observed in only 45% of the mice, and it was less intense. The Adeno.PIA viral vector was unable to raise an anti-P8 I5A response in mice that had been previousty infected with a recombinant adenwirus carrying the fl-galactosidase gene or with a defective adenovirus. We conclude that adenoviral vectors may be very useful for the priming of cytolytic T-cell responses directed against human tumor antigens. Other modes of immunization may be necessary to boost the responses induced with adenoviral vectors.