Children with cerebral palsy who walk with knee flexion during midstance are treated with intramuscular injections of botulinum toxin A (BTX-A) to prevent them from potential deterioration and to improve their mobility. The present study evaluates the effect of this treatment on the muscle activation patterns of the rectus femoris, medial hamstrings and gastrocnemius medialis during gait. Twenty-two children (aged 4-11 years) with cerebral palsy, who walked with knee flexion, were randomly assigned to an intervention group (multilevel BTX-A injections combined with comprehensive rehabilitation) or a control group (usual care). Sagittal and frontal video recordings were made of gait, together with simultaneous surface electromyography recordings of the rectus femoris, medial hamstring and gastrocnemius medialis muscles, before and six weeks after treatment. Abnormal muscle activation patterns were quantified, after gain-normalisation, according to the root mean square difference (RMSD), which is the difference relative to normal patterns. Six weeks after the treatment the RMSD of the gastrocnemius medialis muscles in the intervention group changed significantly, showing a deterioration (p< 0.05). This study demonstrated that BTX-A injections do not result in an improvement in lower limb muscle activation patterns during gait. In spite of this lack of direct effect on muscle activation patterns, the combination of BTX-A injections and comprehensive rehabilitation was effective in improving gait kinematics. © 2010 Elsevier B.V.