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The Drosophila larva is widely used for studies of neuronal development and function, yet little is known about the neuronal basis of locomotion in this model organism. Drosophila larvae crawl over a plain substrate by performing repetitive waves of forward peristalsis alternated by brief episodes of head swinging and turning. To identify sets of central and peripheral neurons required for the spatial or temporal pattern of larval locomotion, we blocked neurotransmitter release from defined populations of neurons by targeted expression of tetanus toxin light chain (TeTxLC) with the GAL4/UAS system. One hundred fifty GAL4 lines were crossed to a UAS-TeTxLC strain and a motion-analysis system was used to identify larvae with abnormal movement patterns. Five lines were selected that show discrete locomotor defects (i.e., increased turning and pausing) and these defects are correlated with diverse sets of central neurons. One line, 4C-GAL4, caused an unusual circling behavior that is correlated with approximately 200 neurons, including dopaminergic and peptidergic interneurons. Expression of TeTxLC in all dopaminergic and serotonergic but not in peptidergic neurons, caused turning deficits that are similar to those of 4C-GAL4/TeTxLC larvae. The results presented here provide a basis for future genetic studies of motor control in the Drosophila larva. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 233–246, 2003 [ABSTRACT FROM AUTHOR]