OBJECTIVE To determine the pharmacodynamic properties of a new oral lyophilisate formulation of desmopressin (in single doses of 30, 60, 120, 240, 360 or 480 µg) in children with known primary nocturnal enuresis (PNE) and thus identify those dosages that could provide a duration of action corresponding to a typical length of night‐time sleep in children with PNE; additional objectives were to determine the safety and tolerability of desmopressin in this population. PATIENTS AND METHODS Children with PNE (mean three or more wet nights/week), aged 6–12 years, were randomized into a double‐blind, placebo‐controlled study. An overhydration technique was used before dosing to suppress endogenous vasopressin production and thereby ensure that any antidiuresis could be attributed to treatment. Dosing with desmopressin or placebo occurred when urinary production was >0.13 mL/min/kg. Urinary volume, osmolality and duration of urinary‐concentrating action (above three threshold levels: 125, 200 and 400 mOsm/kg) were determined as endpoints. RESULTS All 72 participants receiving desmopressin had a pharmacodynamic response to the drug, while there was no change in urinary output in the 12 placebo‐treated patients. There was a clear relationship between desmopressin dose and duration of action and osmolality during action, although the three highest‐dose groups had similar results. The mean duration of action of desmopressin at the lowest osmolality threshold level was 3.6–10.6 h, according to dose; for the highest threshold, the values were 1.3–8.6 h. CONCLUSION Desmopressin, as the oral lyophilisate, causes a marked decrease in urinary output in hydrated children with PNE. A small dose range (120–240 µg) is likely to control diuresis for a period corresponding to a night's sleep (7–11 h) in most children with PNE. However, some patients might require a higher dose to obtain antidiuresis for the complete night.