Abstract The bell-shaped pH rate profile for the reaction of tetrahydrofolic acid with formaldehyde to form 5,10-methylene tetrahydrofolic acid has been shown to result from a change in rate-determining step with changing acidity. In alkaline solution, the rate-determining step is the acid-catalyzed dehydration of hydroxymethyl tetrahydrofolic acid and its conjugate base. This step is subject to general acid catalysis with a Bronsted slope, α, of approximately 0.75. In acid solution, attack of tetrahydrofolic acid on formaldehyde is rate-determining. This step is subject to catalysis by general acids, including the solvated proton, with a Bronsted slope of 0.20. At pH values above 4, the reaction is inhibited by thiols, which combine with formaldehyde in a base-catalyzed reaction to form hemithioacetals. Depending on the reaction conditions, this inhibition can result from a lowering of the equilibrium concentration of formaldehyde or from a trapping by thiol anion of unhydrated formaldehyde, as it is formed from formaldehyde hydrate. The equilibrium constant for hemithioacetal formation from formaldehyde and mercaptoethanol is approximately 620 m-1. Secondary amines such as morpholine and imidazole catalyze the reaction of tetrahydrofolic acid with formaldehyde by nucleophilic catalysis by a pathway which must involve the intermediate formation of cationic imines, [see PDF for structure]