The vaccinia virus HindIII D fragment is 160,060 bp in length and encodes 13 complete open reading frames [Niles et al. (1986) Virology 153, 96-112; S. L. Weinrich and D. E. Hruby (1986). Nucleic Acids Res. 14, 3003-3016]. We have employed a two-step Northern hybridization protocol using single-stranded DNA probes from M13 recombinants in order to identify the mRNA products from the 13 genes. Six of these genes are expressed only at early times after infection; six are transcribed only at late times; one gene is expressed at both early and late times after virus infection. The D11 gene is transcribed into two late mRNA species, one full-length and the other derived from the 3' one-third of the coding sequence. Translation of hybrid-selected mRNA was carried out in an attempt to identify the protein products encoded by each mRNA. Protein products were found for each early gene but translation was successful for only two of the eight late mRNAs. With the completion of the physical map it is apparent that the early and late genes in the HindIII D fragment are arranged in order to minimize potential interference caused by the expression of closely packed viral genes.