Current biosynthetic evidence indicates that methanopterin, a coenzyme involved in C1 metabolism in the methanogenic archaebacteria, is the first example of a naturally occurring, structurally modified folic acid that functions as a coenzyme in cells. Despite these structural modifications, methanopterin functions biochemically in these bacteria in the same way folic acid does in other cells and appears to have taken over all the normal functions of folic acid in these folic acid-lacking cells. In order to understand how and why this change in the structure of the folic acid coenzyme arose, the details of the reactions involved in the biosynthesis of methanopterin have been explored. Recent work on the biosynthesis of the individual units of methanopterin has shown that GTP is a likely precursor to the pterin, the aniline portion of the 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane was shown to arise from p-aminobenzoic acid (via the shikimic acid biosynthetic pathway), and the five-carbon side chain was shown to arise from a pentose (proposed to be ribose on the basis of stereochemistry). Both of the methyl groups of methanopterin are derived from methionine, established by deuterium incorporation from [methyl-2H3]-methionine. Cell extracts of M. volta converted 7,8-H2 neopterin to 7,8-H2-6-hydroxymethylpterin (presumably catalyzed by dihydroneopterin aldolase), and [methylene-2H]-6-hydroxymethylpterin was incorporated into methanopterin to an extent of 29%. [methyl-2H3]-6-[(IRS)hydroxy-ethyl]pterin was incorporated to 8.9%, while 7-methyl pterins were not, indicating the C-9 methyl group is added before pterin coupling with methaniline and the C-7 methyl group later, though cell-free extracts showed condensation before methylation. Methaniline is biosynthesized from p-aminobenzoic acid, confirmed by cell extracts producing 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane and its ribose-P derivative from pAB, ATP, and ribose-P. Condensation of 7,8-H2-6-hydroxymethylpterin with methaniline generates demethylated dihydromethanopterin, which is methylated to methanopterin (confirmed by isolation and methylation experiments with SAM). A pathway for methanopterin biosynthesis is proposed, with reactions supported in methanogenic bacteria. This first reported occurrence of the methylation of a coenzyme further expands our knowledge on nature's use of methylation reactions to control the biochemical reactivity and specificity of biomolecules.