Objective. To determine whether there is a significant and observable behavioural reaction in young children to the entry of vaccine fluid that is separate from the pain of needle puncture. Setting/sample. Twenty-four children, aged 4-6 years, undergoing routine outpatient immunization by intramuscular injection in an Australian paediatric teaching hospital. Measurement. Facial, motor, vocal and verbal reactions, as well as global pain responses were coded by two independent observers as the needle entered the child's arm, and as the vaccine was injected from the syringe. Children and parents used the Faces Pain Scale to give a global rating of needle pain. Results. A distinctive behavioural reaction to the vaccine injection phase of immunization was observed. While children's own needle pain ratings tended to be more extreme than those of their parents, the variance in children's global scores was best predicted by an independent observer's ratings of motor behaviour to needle puncture and vocal response to vaccine injection. Conclusions/implications for practice. A significant pain-related behavioural reaction may be observed in relation to the vaccine injection component of routine immunization. Preparation of children for routine immunization should not focus merely on pain due to needle puncture, but also on potential sensations associated with vaccine injection.