The structures of the modified folates present in Pyrococcus furiosus have been determined. This was accomplished largely by the characterization of the arylamines resulting from the air oxidative cleavage of the reduced modified folates present in these cells, using both chemical and enzymatic methods. Cell extracts separated on DEAE-Sephadex columns showed one major peak containing the arylamines derived from the modified folates. These arylamines were not retained on the DEAE-Sephadex columns, indicating that they contained no net negative charge. Purification of the azo dye derivatives of these arylamines on a Bio-Gel P-6 column showed the presence of three different compounds (compounds 1, 2, and 3) in an average amount of 4.1, 7.6, and 22 nmol/g dry weight of cells, respectively. Each of these compounds readily underwent mild acid hydrolysis (0.1 M HCl, 110 degrees C, 1 min) to produce the azo dye derivative of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane (pAPT). The structure and stereochemistry (ribo) of the pAPT was the same as the pAPT present in methanopterin. In addition, compounds 1, 2, and 3 were each shown to contain 1 mol equiv of ribose and 1, 2, and 3 mol equiv of N-acetylglucosamine (gluNAc), respectively, and were designated as the azo dye derivatives of pAPT-ribose-gluNAc, pAPT-ribose-(gluNAc)2, and pAPT-ribose-(gluNAc)3. Each of these compounds was readily cleaved to the azo dye derivative of pAPT-ribose by the enzymatic action of beta-N-acetylglucosaminidase, indicating that all the gluNAc residues were beta-linked.(ABSTRACT TRUNCATED AT 250 WORDS)