We have previously shown that the myocardium is a target tissue for estrogen. Here, we have identified rapid non-nuclear estrogen effects on the expression of the early growth response gene-1 (Egr-1) in cardiomyocytes. Egr-1 mRNA and protein were rapidly and strongly induced by estrogen in an estrogen receptor-dependent manner via the extracellular signal-regulated kinase, ERK1/2. A promoter analysis study of a 1.2-kilobase Egr-1 promoter fragment revealed that the serum response elements (SREs) but not the estrogen response elements or AP-1 sites are responsible for Egr-1 induction by estrogen, identifying a novel mechanism of estrogen receptor-dependent gene activation in the myocardium. Both estrogen receptor-alpha and -beta induced the Egr-1 promoter via the SREs as well as an artificial promoter consisting of only five SREs in cardiomyocytes. Electrophoretic mobility shift assays showed that a protein complex containing serum response factor or an antigenically related protein was recruited to the SREs by estrogen treatment of primary cardiomyocytes. The recruitment of the protein complex was inhibited by the specific estrogen receptor antagonist ICI 182,780 as well as the MEK inhibitor PD 98059. Taken together, these results identify SREs as important promoter control elements for an estrogen receptor-dependent mechanism of gene activation in the myocardium.