A multicentre, single dose study enrolled 12 previously treated patients with moderately severe to severe (factor IX [FIX] levels <= 2IU) haemophilia B to assess FIX pharmacokinetics after nonacog alfa administration and to evaluate the impact of length of sampling time on half-life (t(1/2)). After refraining from FIX replacement for four days, patients received 50 IU/kg as an intravenous (IV) infusion over 10 minutes. Blood samples were collected predose and 0.25, 0.5, 1, 3, 6, 9, 24, 50, 72, and 96 h post dose. Tolerability and safety were assessed by monitoring adverse events and were subsequently summary tabulated. FIX activity was measured by a one-stage clotting assay with a lower limit of quantification of 0.010 IU/ml, and inhibitors to FIX were measured using the Bethesda assay. Pharmacokinetic parameters were calculated by noncompartmental analysis and were descriptively summarised. Half-life estimates were calculated first using all avail able data, then excluding 96-h observations (truncated at 72 h) and, finally, excluding both 72 and 96-h observations (truncated at 50 h). No patient was positive for FIX inhibitors. No treatment-emergent adverse events were reported. Prolonging the duration of the sample collection to 96 h resulted in a terminal t(1/2) estimate of 39.6 7.4 h in the eight patients aged 18 years and older, which was longer than the estimates obtained using shorter periods of observation: 29.6 5.5 h (truncated at 72 h) and 27.2 7.0 h (truncated at 50 h). To accurately assess an adult patient's th, sampling should be continued for at least 96 h.