Transcription of the LH subunit genes is stimulated by GnRH and may be modulated physiologically by steroids such as 17beta-estradiol (E). We found that E treatment amplified GnRH stimulation of the rat LHbeta and alpha-subunit promoters, and expression of the endogenous mRNA, in LbetaT2 gonadotrope cells 2- to 5-fold above GnRH alone. We examined gene expression in LbetaT2 cells after E and/or GnRH treatment, and found that E suppressed expression of transcription factor Zfhx1a, and enhanced GnRH stimulation of Egr-1 mRNA and protein. E effects were abolished in the presence of antiestrogen. Egr-1 is critical for LHbeta expression; however, the role of Zfhx1a, which binds to E-box sequences, was untested. We found E-box motifs in both the rat LHbeta (-381, -182, and -15 bp) and alpha-subunit (-292, -64, -58 bp) promoters. Zfhx1a overexpression suppressed basal and GnRH-stimulated activity of both promoters. Mutation of the alpha-subunit promoter E boxes at either -64 or -58 bp eliminated Zfhx1a suppression, whereas mutation of the -292 bp E box had no effect. Gel shift assays demonstrated that Zfhx1a bound to the -64 and -58, but not -292, bp E-box DNA. Similarly, mutation of LHbeta promoter E boxes at either -381 or -182, but not -15, bp reduced Zfhx1a suppression, correlating with binding of Zfhx1a. The -381 bp LHbeta E box overlaps with an Sp1 binding site in the distal GnRH-stimulatory region, and increased Sp1 expression overcame Zfhx1a suppression. Thus, one mechanism by which E may enhance GnRH-stimulated LH subunit promoter activity is through regulation of both activators and suppressors of transcription.