The possible regulatory role of tetrahydrobiopterin (BH 4 ) in Type III nitric oxide synthase (NOS III) activity of human placentae from first trimester, term and pre-eclamptic pregnancies was investigated. In homogenates of first-trimester or term placentae, BH 4 stimulated NOS III activity up to 2.5-fold in a concentration dependent manner from 20n m to 1μm BH 4 , and half-maximal stimulation (EC 50 ) was observed at 100–110n m. No significant further stimulation was detectable over an extended concentration range from 1μm to 50μm BH 4 . NOS III present in microsomal and gel-filtered cytosol fractions exhibited similar BH 4 -activation patterns, with an identical EC 50 value of 50n m. Remarkably, tissue concentrations of BH 4 showed a marked decrease in term placentae (57±23n m, mean±s.d., n=26) relative to first-trimester placentae (189±79n m, mean±s.d.,n =17), suggesting that alterations in cellular BH 4 concentrations may play a more significant role in the regulation of NOS III activity in late pregnancy. Placental homogenates from 10 pre-eclamptic pregnancies exhibited two distinct types of response to BH 4 . In seven placental homogenates, addition of physiological concentrations of BH 4 (20n m to 1μm) elicited no increase whatsoever in basal NOS III activity, and only high BH 4 concentrations (50μm) caused notable stimulation (BH 4 resistant group). In contrast, in three of 10 placental homogenates both physiological and 50μm BH 4 concentrations stimulated NOS III to levels similar to that of normal placentae (BH 4 responsive group). There were no appreciable differences in the clinical presentation of pre-eclampsia between the two groups. Importantly, BH 4 concentrations in pre-eclamptic placentae were comparable with those of normal, control placentae. Taken together, the observations suggest that BH 4 controls NOS III activity in the human placenta, and a defect in BH 4 regulation of NOS III may contribute to the development of pre-eclampsia. A model implicating the malfunction of placental NOS III rather than its actual tissue level in the pathogenesis of pre-eclampsia is discussed.