Hydroxyurea therapy reduces the rates of vaso‐occlusive crisis in patients with sickle cell anaemia and recent data suggest that hydroxyurea treatment can generate nitric oxide (NO). Nitric oxide has been proposed as a novel therapy for sickle cell disease via a number of pathways. We therefore sought to determine whether hydroxyurea has NO donor properties in patients with sickle cell anaemia and explore potential mechanisms by which NO production could be therapeutic. Venous blood was collected from 19 fasting sickle cell anaemia patients, on chronic hydroxyurea therapy, at baseline and 2 and 4 h after a single morning dose of hydroxyurea, as well as 10 patients not taking hydroxyurea. The plasma and red cell NO reaction products nitrate, nitrite and nitrosylated‐ haemoglobin were measured using ozone‐based chemiluminescent assays (using vanadium, KI and I₃^– reductants respectively). Consistent with NO release from hydroxyurea, baseline levels of total nitrosylated haemoglobin increased from 300 nmol/l to 500 nmol/l (P = 0·01). Plasma nitrate and nitrite levels also significantly increased with peak levels observed at 2 h. Glutathionyl–haemoglobin levels were unchanged, while plasma secretory vascular cellular adhesion molecule‐1 levels were reduced in patients taking hydroxyurea (419 ± 40 ng/ml) compared with control patients with sickle cell anaemia (653 ± 55 ng/ml; P = 0·003), and were inversely correlated with fetal haemoglobin levels (r = −0·72; P = 0·002). These results demonstrate that hydroxyurea therapy is associated with the intravascular and intraerythrocytic generation of NO. The role of NO in the induction of fetal haemoglobin and possible synergy between NO donor therapy and classic cytostatic and differentiating medications should be explored.