Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylation controls a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated on a given protein. This is often a nontrivial task. In addition to the difficulties involved in purifying sufficient amounts of any given protein, most phosphoproteins contain multiple, substoichiometric sites of phosphorylation. In this paper, we describe substantial improvements made to our previously reported multidimensional electrospray MS-based phosphopeptide mapping technique that have resulted in a 20-fold increase in sensitivity for the overall process. Chief among these improvements are the incorporation of capillary chromatography and a microionspray source for the mass spectrometer into the first dimension of the analysis. In the first dimension of the process, phosphopeptides present in the proteolytic digest of a protein are selectively detected and collected into fractions during on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ion scanning. The relative merits and limitations of these two techniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the method under the new experimental conditions makes it suitable for phosphorylation mapping (from selective detection through sequencing) on gel-separated phosphoproteins where the level of phosphorylation at any given site is <200 fmol. Furthermore, this method detects serine, threonine, and tyrosine phosphorylation equally well. We have successfully employed this new configuration to map 11 in vivo sites of phosphorylation on the Saccharomyces cerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate for PKA. In addition, four peptides containing cdk sites and the autophosphorylation site at Tyr530 were found to be phosphorylated. Because the first dimension of this method generates a phosphorylation profile that can be used for a semiquantitative evaluation of site specific phosphoxylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a protein in response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detect a change in phosphorylation stoichiometry on the motor protein myosin-V in response to treatment with either mitotic or interphase Xenopus egg extracts and to identify the single functionally significant phosphorylation site that regulates myosin-V cargo binding.