Tight regulation of luteinizing hormone-beta subunit (LHbeta) expression is critical for differentiation and maturation of mammalian sexual organs and reproductive function. Two transcription factors, steroidogenic factor-1 (SF-1) and early growth response-1 (Egr-1), play a central role in activating LHbeta promoter, and the synergy between these two factors is essential in mediating gonadotropin-releasing hormone stimulation of LHbeta promoter. Here we demonstrate that the transcriptional co-activator p300 regulates this synergy. Overexpression of p300 results in strong stimulation of LHbeta promoter but only in the presence of both SF-1 and Egr-1, and not in the presence of other Egr proteins. Mutation of the binding sites for either SF-1 or Egr-1 completely abolishes the synergy between these two factors, as well as the influence of p300. Importantly, LHbeta promoter is precipitated using p300 antibodies in a chromatin immunoprecipitation assay with LbetaT2 gonadotropes, and this effect is enhanced by gonadotropin-releasing hormone. The influence of p300 on LHbeta promoter is potentiated by steroid receptor co-activator, as well as by E1A proteins, and attenuated by Smad nuclear interacting protein 1. Taken together, these results suggest that p300 is recruited to LHbeta promoter where it coordinates the functional synergy between SF-1 and Egr-1.