A variety of chemically modified oligonucleotides have been studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) in the negative ion mode. These include oligonucleotides containing modified bases, such as uracil glycol, bromoguanine, O6-butylguanine, as well as oligonucleotides in which the phosphodiester groups had been replaced by other functional groups, such as phosphorothioates. With the linear TOF mass spectrometer, there is no or very little fragmentation observed, and the determination of the molecular weight by MALDI-TOFMS offers a convenient way for identifying/confirming the presence of the modification. With internal calibration, a mass accuracy of 0.01% can be achieved. Such mass accuracy makes it possible to directly differentiate a small uridine-containing oligonucleotide from its cytidine-containing analogue. Because of factors such as sample inhomogeneity, laser output fluctuation, and the dynamic range of the detector, quantitation by MALDI-TOFMS has been difficult. Nevertheless, semiquantitative information can be obtained for those analytes that are closely related in structure. Monitoring the products of the synthesis of monophosphorothioated oligoribonucleotide 16-mers by MALDI-TOFMS revealed that the sulfur atom in the phosphorothioate group can be replaced by an oxygen atom during the succeeding introduction of phosphodiester groups. The earlier the phosphorothioate group is introduced during the synthesis of the 16-mer, the greater is the extent of sulfur to oxygen replacement.