Although proteins phosphorylated on tyrosine residues can be enriched by immunoprecipitation with anti-phosphotyrosine antibodies, it has been difficult to identify proteins that are phosphorylated on serine/threonine residues because of lack of immunoprecipitating antibodies. In this report, we describe several antibodies that recognize phosphoserine/phosphothreonine-containing proteins by Western blotting. Importantly, these antibodies can be used to enrich for proteins phosphorylated on serine/threonine residues by immunoprecipitation, as well. Using these antibodies, we have immunoprecipitated proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine/threonine-phosphorylated proteins including drebrin 1, alpha-actinin 4, and filamin-1. We also identified a protein, poly(A)-binding protein 2, which was previously not known to be phosphorylated, in addition to a novel protein without any obvious domains that we designate as Frigg. Frigg is widely expressed and was demonstrated to be a protein kinase A substrate in vitro. We identified several in vivo phosphorylation sites by tandem mass spectrometry using Frigg protein immunoprecipitated from cells. Our method should be applicable as a generic strategy for enrichment and identification of serine/threonine-phosphorylated substrates in signal transduction pathways.