A novel pyrenyl-containing phosphoramidite reagent, N-[4-(l-pyrenyl)butyryl]-O1-(4, 4'-dimethoxytrityl)- O2-[(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-l, 2-propanediol (5), has been synthesized from 4-(l-pyrenyl)butanoic acid in four steps with the 52% overall yield and used to incorporate pyrene residue(s) into oligonucleotides. Oligonucleotides 6 and 7, bearing one or two pyrenes at the 5'-terminus, have been prepared by means of that reagent, characterized with fluorescence spectra, and successfully used as primers in a polymerase chain reaction. © 1992, American Chemical Society. All rights reserved.