We have established the biosynthetic pathway and the associated genes for the biosynthesis of the 5-(aminomethyl)-3-furanmethanol (F1) moiety of methanofuran in the methanogenic archaeon Methanocaldococcus jannaschii. The recombinant enzyme, derived from the MJ1099 gene, was shown to readily condense glyceraldehyde 3-phosphate (Ga-3P) and dihydroxyacetone-P (DHAP) to form 4-(hydroxymethyl)-2-furancarboxaldehyde phosphate (4-HFC-P). The recombinant purified pyridoxal 5'-phosphate-dependent aminotransferase, derived from the MJ0684 gene, was found to be specific for catalyzing the transamination reaction between 4-HFC-P and [(15)N]alanine to produce [(15)N] 5-(aminomethyl)-3-furanmethanol-P (F1-P) and pyruvate. To confirm these results in cell extracts, we developed sensitive analytical methods for the liquid chromatography-ultraviolet-electrospray ionization mass spectrometry analysis of F1 as a 7-nitrobenzofurazan derivative. This method has allowed for the quantitation of trace amounts of F1 and F1-P in cell extracts and the measurement of the incorporation of stable isotopically labeled precursors into F1. After incubation of cell extracts with [1,2,3-(13)C3]pyruvate and DHAP, 4-([(2)H2]hydroxymethyl)-2-furancarboxylic acid phosphate (4-HFCA-P) or 4-([(2)H2]hydroxymethyl)-2-furancarboxaldehyde phosphate (4-HFC-P) was found to be incorporated into F1-P. 4-HFCA-P and 4-HFC-P were confirmed in cell extracts after removal of the phosphate. The low level of incorporation of [1,2,3-(13)C3]pyruvate into F1-P in these experiments is explained by the fact that the labeled pyruvate must first be converted into Ga-3-P through gluconeogenesis before being incorporated into 4-HFC-P. Cell extracts incubated with 4-HFC-P and a mixture of [(15)N]aspartate, [(15)N]glutamate, and [(15)N]alanine produced [(15)N]F1-P. We also demonstrated that aqueous solutions of methylglyoxal or pyruvate heated with dihydroxyacetone led to the formation of 4-HFC and 4-HFCA, suggesting a possible prebiotic route to this moiety of methanofuran.