Endothelial nitric oxide synthase (NOS) protein and mRNA have been identified and calcium-dependent NOS activity has been measured in human placentae during normal pregnancy. Recently, mRNA and protein for the inducible isoform of NOS have been detected in placentae of women with gestational diabetes. The aim of this study was to determine whether calcium-independent (ciNOS) and/or total (tNOS) NOS activities were increased in placentae obtained after vaginal delivery or Caesarean section from women assigned to the following groups according to standard obstetric criteria: gestational diabetes, diabetes before pregnancy and non-diabetic controls. tNOS and ciNOS were assessed by measuring the conversion of [ 3 H]⌊-arginine to [ 3 H]⌊-citrulline in the three groups. Michaelis–Menten constants ( K m ) and maximum velocities of reaction ( V max ) were calculated using Lineweaver–Burk analysis for tNOS. There were no significant differences in either ciNOS, V max or K m values between any of the three groups (normal, ciNOS 12.7±1.6%, V max 16.6±3.3 pmol⋅min -1 ⋅mg -1 protein, K m 15.30±2.6 µ mol/l; gestational diabetes, ciNOS 15.4±1.4%, V max 14.8±5.2 pmol⋅min -1 ⋅mg -1 protein, K m 10.5±1.7 µ mol/l; diabetes before pregnancy, ciNOS 13.4±1.1%, V max 14.9±3.4 pmol⋅min -1 ⋅mg -1 protein, K m 17.7±2.2 µ mol/l). The presence of macrosomia did not affect tNOS activity in those with diabetes before pregnancy, and glycosylated haemoglobin levels measured between weeks 27 and 39 were not correlated with ciNOS activity. The results from the present study do not provide evidence for increased placental tNOS or ciNOS activities in pregnancies complicated by gestational diabetes or diabetes present before pregnancy.