To assess in suckling lambs the impact of intradermal injection of cetrimide, a quaternary ammonium compound formulated to induce non-surgical mulesing, on some physiological and behavioural indicators of welfare. We allocated 32 suckling lambs (9-11 weeks old) to three groups: (1) control (n = 10), (2) conventional surgical mules (n = 11) and (3) non-surgical mules (n = 11). Non-surgical mulesing was induced by intradermal injection of 4% (w/w) cetrimide + 3% (w/w) polyvinylpyrrolidone in water. Lambs were run in pens of four together with their dams. Haematology, cortisol, beta-endorphin and haptoglobin levels, and rectal temperature were monitored at least daily for the first 7 days after treatment, then weekly until day 28. Body weight was measured weekly and behaviour was measured every 15 min for 12 h on the day of treatment, then on days 1, 2, 4, 6, 12, 21 and 28 following treatment. The intradermal treatment induced local tissue swelling, systemic signs of severe inflammation, including high fever (> 41.0 degrees C) and elevated blood cortisol levels, by 12 h. Rectal temperatures were significantly elevated until 6 days after treatment, cortisol levels were elevated until 4 days after treatment, haptoglobin levels for at least 7 days after treatment and the neutrophil to lymphocyte ratio until 5 days after treatment. Peak cortisol values were comparable in mulesed lambs and lambs receiving the intradermal treatment, whereas the areas under the curves for cortisol and temperature were greater in lambs receiving the intradermal treatment than in mulesed lambs. Beta-endorphin levels were significantly elevated in mulesed sheep at 12 h. There was no effect of intradermal treatment on average daily gain, fibre diameter or beta-endorphin concentration. Mulesed lambs spent 44% of the time in abnormal behaviours (hunched standing, stiff walking, pawing, lateral lying and lying intention) on the day of treatment. On the day after treatment, lambs receiving the intradermal treatment spent 11% of the time (comparable to mulesed lambs) in abnormal behaviours. In comparison, control lambs spent 0.4% of their time in abnormal behaviours on the same day. The welfare of suckling lambs that were non-surgically mulesed by intradermal injection of cetrimide was measurably poorer than control lambs.