The effect of osmium tetroxide oxidation of single-stranded, circular φX174-DNA on its infectivity in E. coli spheroplasts was investigated. Osmium tetroxide predominantly oxidizes thymine to 5,6-dihydroxy-dihydrothymine in DNA and causes only negligible amounts of strand breakage. Products of the 5,6-dihydroxy-dihydrothymine type represent typical lesions introduced into DNA by ionizing radiation. It was found that osmium tetroxide oxidation inactivated the DNA and that at 37% residual infectivity the φX174-genome contained on average 2.8 ring-saturated thymine residues. No significant differences were observed for spheroplasts from wild type E. coli, E. coli polA, E. coli uvrA, E. coli recA, and E. coli recB.