In contrast to the single-stranded circular molecules produced with denatured DNA from other adenoviruses, there was associated with nearly all circular molecules of adenovirus type 18 a visible, duplex projection. These projections had a mean contour length of 0.31 +/- 0.12 mum, equivalent to approximately 3% of genome length. Individual projections ranged in size from 0.1 to 2 mum. Alkaline sucrose gradient purification of single-stranded molecules did not affect formation of these projections, and treatment of a preparation of circular molecules with Neurospora crassa single-strand specific nucleases yielded 0.34 +/- 0.09 mum duplex fragments. Single-stranded circles did not form if a limited number of nucleotides were removed from the 3' ends of native molecules by Escherichia coli exonuclease III digestion prior to denaturation and annealing. In addition, preformed single-stranded circles could be converted to linear molecules by similar treatment. Based on the formation of specific heteroduplex structures when preparations of native DNA were denatured and reannealed and the absence of branches on linear, single-stranded molecules, we conclude that projections are generated by unusually long, inverted terminal repetitions. The repetitious sequences occur in place of rather than in addition to regular sequences. These data provide direct, visual evidence for the arrangement of bases in the inverted terminal repetition of adenovirus DNA.