Column chromatography and spectroscopy have been employed in analyzing pyrimidine derivatives obtained from alkaline-treated 7-methylguanosine (7-meGuo). High performance liquid chromatography (HPLC) revealed that the alkaline generated products consist predominantly of two forms of ring opened 7-methylguanine (rom7Gua) in equal amounts. Material from both Dowex 50 and Sephadex LH-20 columns was readily resolvable into two HPLC peaks. The species in one peak appears to be composed of formylated and that in the other of deformylated rom7Gua. The presence of a deformylated species is supported by the absence of radioactivity in one of the two peaks obtained when ring opened [8-14C]-guanosine was analyzed by HPLC. The formylated species was retained on the liquid chromatography column for 8 min with a 3% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent and for 6 min with a 6% methanol, 0.01 N NH4H2PO4 (pH 5.1) solvent system; the deformylated species was retained for 6.3 min with the first solvent and 4.5 min with the second solvent. Subsequent to Dowex 50 chromatography in an ammonium formate solvent, abut 90% of the material was formylated. When stored at 24 degrees C for 72 h in a solvent without formate ions, the material was shown by HPLC to consist of equal amounts of the formylated and deformylated species. These results indicate that the two species of rom7Gua are in equilibrium. The rom7Gua excised from DNA by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species.