Herpes simplex virus 1 (HSV-1) specifies in the infected cell a set of polypeptides (alpha) whose mRNAs are made in the absence of protein synthesis. The individual mRNAs specifying alpha polypeptides 0, 4, 22, and 27 were purified by hybridization to strand-separated or total HSV-1 DNA fragments cloned in pBR322 plasmids, translated in vitro to verify their specificity, then mapped by hybridization to separated strands and digests of cloned DNA fragments. To map the transcription initiation sites, chemically decapped individual mRNAs were recapped with [alpha-32P]GTP and vaccinia virus guanylyltransferase and hybridized to digests of the cloned DNA fragments. Each of the labeled 5' termini hybridized to a specific site, two on one strand, and two on the other. The 5' ends of the transcripts do not share sequence homology, suggesting that they are transcribed from independent promoters.