In herpes simplex virus 1, the five alpha genes are induced by alpha-transinducing factor (alpha TIF; VP16), a virion protein, acting in concert with Oct-1 and other cellular proteins on a cis-acting site in the promoter domain of alpha genes. Because alpha TIF is an essential virion protein, its function as an inducer can best be evaluated only by mutating the cis-acting site. Earlier we reported on a series of 17 mutations in and around the cis-acting site of a 275-bp alpha 27 promoter fused to a reporter gene and recombined into the viral genome. These recombinant viruses were tested in Vero cells in the presence of cycloheximide, and we demonstrated that mutations in the sequence required for Oct-1 binding abolished transactivation whereas mutations in the alpha TIF-dependent GARAT sequence decreased but did not abolish transactivation. We now report that (i) in limited-passage human embryonic lung cells, alpha gene expression from promoters mutated in the GARAT sequences is often higher and more variable than in Vero cells, (ii) in the absence of cycloheximide, the mutant viruses show less significant impairment of reporter gene expression, (iii) Oct-1 can bind either to the overlapping octamer element or to various TAATGARAT sequences with differing degrees of binding strength and these relative binding levels correlate well with levels of gene expression observed in infected cells, (iv) in the cis-acting site upstream of the alpha 4 gene, no degenerate overlapping Oct-1 sequence exists, and therefore in this instance Oct-1 must be binding directly to the TAATGARAT sequence, (v) extension of the alpha 27 promoter by an additional 1,334 bp results in much higher expression of the reporter gene as a result of additional upstream cis-acting sites, and (vi) obliteration of the most proximal Oct-1 binding element within the 275-bp promoter dramatically reduces gene expression even in the presence of the additional upstream cis-acting sites.