We have determined the DNA sequence of the herpes simplex virus type 1 (HSV-1) gene encoding the immediate early protein IE110, which is involved in transcriptional activation of later virus genes. The locations of the 5' and 3' termini of IE110 mRNA together with the positions of two introns, were identified. Examination of the DNA sequence suggested that translation starts at the first ATG after the 5' terminus of the mRNA, and that both introns occur in protein-coding sequence. The predicted IE110 polypeptide contains 775 amino acids, and has a molecular weight of 78452. It contains a cysteine-rich region resembling regions found in several proteins which interact functionally with DNA. An antiserum was raised to the predicted C terminal amino acid sequence of the IE110 polypeptide and was shown to immunoprecipitate the native protein from HSV-1-infected cell extracts. The functional importance of regions of the protein was evaluated by construction of frameshift and deletion mutants of a plasmid-borne IE110 gene. The mutants were tested for IE110 function by short-term transfection assays, and the results were correlated with the DNA sequence and RNA mapping studies.