We have revisited the direct analysis experiments reported by Tomer and co-workers in the MALDI-TOFMS analysis of phosphopeptide-loaded immobilized metal ion affinity chromatography (IMAC) beads (Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282). The results described herein provide no evidence to support a laser-induced direct desorption of phosphopeptides chelated on IMAC beads. However, we have established that solubilization of mono-phosphopeptides from their immobilized Fe3+-NTA chelates does occur effectively in solutions containing certain MALDI matrices. Particularly effective is 2,5-dihydroxybenzoic acid (2,5-DHB), which apparently forms a stronger chelation complex with Fe3+-NTA than mono-phosphopeptides. With regard to the disparity observed between the low pH value of MALDI matrices (saturated 2,5-DHB(aq) approximately pH 2) and the high pH values of conventional IMAC eluents (typically above pH 7), we have also investigated the influence of eluent pH on the recovery of phosphopeptides from IMAC media. Finally, we have confirmed the importance of employing ammonium dihydrogen phosphate as buffer to achieve effective liberation of mono- and all poly-phosphopeptide species from Fe3+-NTA IMAC resin.