Recent studies have shown that p21-activated kinase 1 (Pak1) phosphorylates estrogen receptor-alpha (ER alpha) at Ser 305 and also promotes its transactivation function. Here, we sought to investigate whether substitution of serine 305 in ER with glutamic acid (ER alpha-S305E), which mimics the phosphorylation state, would influence the status of ER-target genes. To explore this possibility, we generated clones overexpressing ER alpha-S305E in ER-negative MDA-MB-231 cells and analyzed the status of ER-regulated genes using a gene array. Results indicated that the expression of ER alpha-S305E is sufficient to upregulate the expression of a few but not all ER-regulated genes, i.e., cyclin D1 and zinc finger protein 147 (estrogen-responsive finger protein), while there was no significant change in the expression of remaining genes on the array. In addition, we found an increased expression as well as nuclear accumulation of cyclin D1 protein in MDA-MB-231 cells expressing ER alpha-S305E as compared to the level of cyclin D1 in MDA-MB-231 cells expressing WT-ER alpha or pcDNA. Furthermore, ER alpha-S305E, but not mutation of ER alpha-S305 to alanine, enhanced the cyclin D1 promoter activity. These findings suggest that ER alpha activation at S305 is sufficient to upregulate the expression of cyclin D1, an ER-regulated gene that is implicated in the progression of breast cancer. Phosphorylation of ER alpha by Pak1 or its upstream regulators could upregulate the expression of a subset of ER-target genes in a ligand-independent manner and hence, might contribute toward the development of hormone independence in breast cancer cells.