Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from lysates of infected cells as detected by a gel electrophoresis binding assay and that monoclonal antibody to alpha 4 protein reduced the electrophoretic mobility of the complex of labeled DNA and protein from infected cells. In this study we identified one monoclonal antibody, H950, from a panel of monoclonal antibodies to the alpha 4 protein that blocks the formation of specific infected cell complexes with labeled DNA fragments containing promoter and regulatory domains of alpha genes. We also report the nucleotide sequence of the alpha 0 promoter domain protected from exonuclease III digestion by alpha 4 protein in the absence of H950 monoclonal antibody but not in its presence. In addition, we identified a 59-base-pair sequence from the regulatory domain of the alpha 4 gene that binds alpha 4 protein. Deletion clones of this fragment localize sequence elements required for formation of the alpha 4 protein-DNA complex. Furthermore, deletion of the in vitro binding site of the SP1 transcription factor from the 59-base-pair fragment did not affect the formation of the alpha 4 protein-DNA complex.