An inverted terminal repetition was observed in DNA molecules extracted from vaccinia virus. The repeated sequence was visualized by (i) nicking the hairpin loops present of the ends of vaccinia virus DNA, (ii) separating the strands of DNA by alkali denaturation, (iii) allowing the single strands to self-anneal, and (iv) examining the DNA with an electron microscope. Single-stranded circular molecules, each of which contained a duplex projection (3.54 +/- 0.12 micron) representing the terminal repetition, readily formed. Similar size projections were also seen in heteroduplex structures formed by crosshybridization of the separated strands of the two terminal HindIII restriction fragments. Based on contour length measurements and the electrophoretic mobility of the isolated inverted terminal repetition, a molecular weight of approximately 6.9 X 10(6), equivalent to about 10,500 nucleotide base pairs, was estimated. Evidence was obtained from DNA-RNA hybridization studies that the terminal repetition is transcribed.