Earlier studies concerning gamma 1 gene regulation by the alpha 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1), in stably transformed cell lines, reported conflicting results, i.e., alpha 4 protein positively regulated the gamma 1 gB gene in alpha 4/gB cells, while it negatively regulated the gamma 1 gD gene in alpha 4/BJ cells. Both cell lines were derived from a common parental cell line alpha 4/c 113 that contains 1 copy of the alpha 4 gene, and the only apparent difference between them was the relative copy number of the gB and gD sequences (1 and 30-50, respectively) resident in the cell genome. We investigated this disparity by constructing a cell line (BA 4) that contains one copy each of the alpha 4 and gamma 1 gD sequences, by fusion of alpha 4/c 113 and BJt cells, containing and expressing respectively 1 copy of the alpha 4 and gD genes. BA 4 cells constitutively expressed both the alpha 4, gD genes inherited from the parental cell lines (alpha 4/c 113 and BJt). In BA 4 cells that alpha 4 protein positively regulates the gD gene as evidenced from (i) higher levels of gD expression than the parental BJt cells lacking the alpha 4 gene, and (ii) significant decrease in gD expression under conditions that render the alpha 4 protein produced in BA 4 cells non-functional. In addition the gamma 2gG gene contained within the DNA fragment encoding the gD gene, is also expressed in BA 4 cells. On the basis of these data, we propose that gamma gene regulation by the alpha 4 protein is affected by the relative copy number of these genes, resident in the cell genome.