Fluorescent nucleoside analogs provide a means to study DNA interactive systems through direct measurement of fluorescence properties. As integrated parts of DNA, these probes provide opportunities for monitoring subtle changes in DNA structure as it meets and reacts with other molecules. This protocol describes modifications to standard DNA synthesis to efficiently use smaller volumes of the probe phosphoramidite, purification of pteridine-containing sequences and a deprotection procedure specific for 6MI-containing sequences. Yields for probe incorporation in DNA synthesis are comparable to those for standard phosphoramidites. Examples of the fluorescence signals one can expect are described. Automated synthesis, which is dependent on the length of the sequence, takes about 4–5 h for a 20-mer. The deprotection of 6MI-containing sequences takes approximately 6–7 h before the standard ammonium hydroxide overnight incubation. Purification through polyacrylamide gels, electroelution and ethanol precipitation can be accomplished in 6–8 h.