Viruses and other nucleoprotein complexes are inactivated on exposure to white light in the presence of acridine and related dyes. The mechanism is thought to involve generation of singlet oxygen or related species, but the actual molecular targets of the inactivating event have not been well defined. We have re-examined the mechanism of dye-sensitized photoinactivation taking advantage of the well characterized bacteriophage P22. Though the inactivated phage absorb to their host cells, the cells are not killed and genetic markers cannot be rescued from the inactivated phage. These observations indicate that the chromosome is not injected into the host cell. However, the DNA of the damaged particles shows no evidence of double-stranded breaks or crosslinking. The DNA injection process of P22 requires three particle-associated proteins, the products of genes 7, 16 and 20. Gp16, which can act in trans during injection, is inactivated in the killed particles. Sodium dodecyl sulfate/polyacrylamide gel analysis reveals that gp16, gp7 and gp20 are progressively covalently damaged during photoinactivation. However, this damage does not occur in particles lacking DNA, indicating that it is DNA-mediated. Similar findings were obtained with acridine orange, acridine yellow, proflavin and acriflavin. These results indicate that the actual targets for inactivation are the DNA injection proteins, and that the lethal events represent absorption of photons by acridine molecules stacked in a region of DNA closely associated with the injection proteins.