Reported here is how modified oligonucleotides with increased affinity for DNA or RNA target strands can be selected from small combinatorial libraries via spectrometrically monitored selection experiments (SMOSE). The extent to which target strands retard the degradation of 5'-acyl-, 5'-aminoacyl-, and 5'-dipeptidyl-oligodeoxyribonucleotides by phosphodiesterase I (EC 18.104.22.168) was measured via quantitative MALDI-TOF mass spectrometry. Oligonucleotide hybrids were prepared on solid support, and nuclease selections were performed with up to 10 modified oligonucleotides in one solution. The mass spectrometrically monitored experiments required between 120 and 300 pmol of each modified oligonucleotide, depending on whether HPLC-purified or crude compounds were employed. Data acquisition and analysis were optimized to proceed in semiautomated fashion, and functions correcting for incomplete degradation during the monitoring time were developed. Integration of the degradation kinetics provided "protection factors" that correlate well with melting points obtained with traditional UV melting curves employing single, pure compounds. Among the components of the five libraries tested, three were found to contain 5'-substituents that strongly stabilize Watson--Crick duplexes. Selecting and optimizing modified oligonucleotides via monitored nuclease assays may offer a more efficient way to search for new antisense agents, hybridization probes, and biochemical tools.