Activated estrogen receptor (ERalpha) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the N-terminal domain (NTD) contributes to ERalpha activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ERalpha residues was limited to protein artificially overexpressed in transfected cell lines. We report mass spectrometric methods that have allowed the identification of a new site within the NTD of ERalpha isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nano-LC-ESI-MS/MS (Q-STAR, MDS Sciex) and vMALDI-MS(n) (Finnigan LTQ, Thermo-Electron) identified peptides containing 8 of 14 serine residues within the NTD, one being partially phosphorylated Ser-167, known but not previously reported by MS. Chymotrypsin digestion revealed other known sites at Ser-102/104/106 and 118. Tandem methods developed for the peptide containing Ser-118 and the use of hypothesis-driven experiments--i.e., the assumption that an intact phosphopeptide showing no molecular ion might yield fragment ions including loss of phosphoric acid in vMALDI-MS/MS--allowed the identification of a novel site at Ser-154. Quantitation by selected reaction monitoring demonstrated 6-fold and 2.5-fold increases in Ser-154 phosphorylation in estradiol- and EGF-treated cells, respectively, compared to controls, confirmed by immunoblotting with a novel rabbit polyclonal antibody. Thus, the protein isolation and MS strategies described here can facilitate discovery of novel phosphorylation sites within low abundance, clinically important cancer targets like ERalpha, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer.