Single 13C6-labelled doses of pteroylmonoglutamic acid (PteGlu; 634 nmol) or 5-formyltetrahydrofolic acid (431–569 nmol) were given to fasted adult volunteers, and the rise in total and 13C-labelled plasma 5-methyltetrahydrofolic acid metabolite monitored over 8 h by HPLC and liquid chromatography–MS. The dose-adjusted area under the curve (AUC) for total (labelled plus unlabelled) plasma 5-methyltetrahydrofolic acid following a 5-formyltetrahydrofolic acid test dose was 155 % that obtained following a PteGlu test dose. Surprisingly, an average 60 and 40 % of the total plasma 5-methyltetrahydrofolic acid response to [13C6]PteGlu and [13C6]5-formyltetrahydrofolic acid, respectively, was unlabelled; an observation never before reported. Short-term kinetics of plasma [13C6]5-methyltetrahydrofolic acid showed a slower initial rate of increase in plasma concentration and longer time to peak following an oral dose of [13C6]PteGlu compared with that for an oral dose of [13C6]5-formyltetrahydrofolic acid, while the [13C6]5-methyltetrahydrofolic acid AUC for [13C6]5-formyltetrahydrofolic acid was 221 % that for [13C6]PteGlu. These data indicate that PteGlu and 5-formyltetrahydrofolic acid, which are thought to be well absorbed (about 90 %) at physiological doses, exhibit dramatically different rates and patterns of plasma response. A limitation in the rate of reduction of PteGlu before methylation could result in slower mucosal transfer of [13C6]5-methyltetrahydrofolic acid derived from [13C6]PteGlu into the plasma. This, when coupled with an observed similar plasma clearance rate for [13C6]5-methyltetrahydrofolic acid metabolite derived from either folate test dose, would yield a comparatively smaller AUC. These findings suggest potential problems in interpretation of absorption studies using unlabelled or labelled folates where the rate of increase, the maximum increase, or the AUC, of plasma folate is employed for test foods (mainly reduced folates) v. a ‘reference dose’ of PteGlu.