The 5′ ends of two early herpes simplex virus type 1 mRNAs have been identified by nuclease S1 and exonuclease VII analysis using cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region between map coordinates 0.56 and 0.60, are unspliced and share a 3′ terminus. Genomic DNA at the 5′ ends has been sequenced and the 5′ termini have been located on the virus DNA sequence. The DNA sequence has revealed signals involved in the initiation of transcription of both mRNAs, and the 5′ end of the 1.2 kb mRNA is encoded within the internal sequences of the 5.0 kb mRNA. The probable translational initiation codons for the polypeptides specified by these mRNAs have been identified, and the results indicate that the coding regions of the two mRNAs do not overlap.