The colinearity of RNA transcripts with the vaccinia virus genome was investigated. Cytoplasmic RNA from infected cells was annealed to a cloned DNA segment that extended from 9 to 15.6 kilobase pairs from the left end of the genome and contained approximately 800 base pairs of the inverted terminal repetition (ITR). Remaining unhybridized single strands of DNA were digested with nuclease S1, and the lengths of the protected DNA fragments were determined by agarose gel electrophoresis under neutral and alkaline conditions. Uniformly 32P-labeled cloned DNA insert, separated recombinant DNA strands, and smaller restriction fragments, as well as 3' and 5' end-labeled DNA, were employed to map five early RNAs and one late RNA. One of the early RNAs hybridized to sequences within the ITR, and the other four hybridized to sequences proximal to the ITR. The late RNA was initiated proximal to the ITR but extended into it. Interestingly, the 3' portion of this late RNA was complementary to the early RNA transcribed from the opposite strand of the ITR. From a comparison of the lengths of the protected DNA fragments on neutral and alkaline gels, all except the complementary early and late RNAs appeared to be colinear with the genome. Although the anomalous nuclease S1 data obtained with the latter RNAs mimicked splicing, they were shown by DNA-DNA hybridization to result from a small deletion or rearrangement within the ITR. Thus far, no true examples of spliced vaccinia virus RNAs have been found.