Transcriptional activation of the prolactin and growth hormone genes, occurring in a cell-specific fashion, requires short-range synergistic interactions between the pituitary-specific POU domain factor Pit-1 and other transcription factors, particularly nuclear receptors. Unexpectedly, we find that these events involve the gene-specific use of alternative Pit-1 synergy domains. Synergistic activation of the prolactin gene by Pit-1 and the estrogen receptor requires a Pit-1 amino-terminal 25-amino-acid domain that is not required for analogous synergistic activation of the growth hormone promoter. The action of this Pit-1 synergy domain is dependent on the presence of two of three tyrosine residues spaced by 6 amino acids and can be replaced by a comparable tyrosine-dependent trans-activation domain of an unrelated transcription factor (hLEF). The gene-specific utilization of this tyrosine-dependent synergy domain is conferred by specific Pit-1 DNA-binding sites that determine whether Pit-1 binds as a monomer or a dimer. Thus, the critical DNA site in the prolactin enhancer, where this domain is required, binds Pit-1 as a monomer, whereas the Pit-1 sites in the growth hormone gene, which do not utilize this synergy domain, bind Pit-1 as a dimer. The finding that the sequence of specific DNA sites dictates alternative Pit-1 synergy domain utilization based on monomeric or dimeric binding suggests an additional regulatory strategy for differential target gene activation in distinct cell types.