Mass spectrometric characterization of engineered proteins has been examined using bovine recombinant Acyl-CoA-Binding Protein (rACBP), [15N]-labeled rACBP, and a number of sequence variants of ACBP produced by site-directed mutagenesis. The mass spectrometric techniques include ESIMS and MALDIMS for analysis of the intact protein. Peptide maps have been obtained either by direct analysis of enzymatically derived mixtures by PDMS, ESIMS, and MALDIMS or by off- and on-line HPLC-mass spectrometry. ESIMS was found to be most accurate for analysis of intact proteins. The best sequence coverage in mapping was obtained by LC-ESIMS and by direct mixture analysis by MALDIMS. The latter technique was favorable in terms of sensitivity and speed. A general strategy for mass spectrometric characterization of engineered proteins is suggested.