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Desert locusts (Schistocerca gregaria) can undergo a profound transformation between solitarious and gregarious forms, which involves widespread changes in behaviour, physiology and morphology. This phase change is triggered by the presence or absence of other locusts and occurs over a timescale ranging from hours, for some behaviours to change, to generations, for full morphological transformation. The neuro-hormonal mechanisms that drive and accompany phase change in either direction remain unknown. We have used high-performance liquid chromatography (HPLC) to compare amounts of 13 different potential neurotransmitters and/or neuromodulators in the central nervous systems of final instar locust nymphs undergoing phase transition and between long-term solitarious and gregarious adults. Long-term gregarious and solitarious locust nymphs differed in 11 of the 13 substances analysed: eight increased in both the brain and thoracic nerve cord (including glutamate, GABA, dopamine and serotonin), whereas three decreased (acetylcholine, tyramine and citrulline). Adult locusts of both extreme phases were similarly different. Isolating larval gregarious locusts led to rapid changes in seven chemicals equal to or even exceeding the differences seen between long-term solitarious and gregarious animals. Crowding larval solitarious locusts led to rapid changes in six chemicals towards gregarious values within the first 4 h (by which time gregarious behaviours are already being expressed), before returning to nearer long-term solitarious values 24 h later. Serotonin in the thoracic ganglia, however, did not follow this trend, but showed a ninefold increase after a 4 h period of crowding. After crowding solitarious nymphs for a whole larval stadium, the amounts of all chemicals, except octopamine, were similar to those of long-term gregarious locusts. Our data show that changes in levels of neuroactive substances are widespread in the central nervous system and reflect the time course of behavioural and physiological phase change.