The dorsal longitudinal flight muscle (DLM) of Drosophila is composed of six muscle fibers (DLM1-6 from ventral to dorsal) and innervated by five motor neurons (MNs), DLM1-4 being innervated singly by MN1-4, and DLM5 and 6 being jointly innervated by MN5. This study identifies and describes the five motor neurons that innervate these six muscle fibers. Horseradish peroxidase (HRP) applied intracellularly to single identified DLM fibers resulted in the labeling of the single motor neuron innervating that muscle fiber by retrograde transsynaptic transport. This method allowed positive identification of the motor neuron innervating a particular muscle fiber, since only the innervating neuron was labeled. The axonal pathway, soma, and dendritic distribution of each labeled motor neuron was traced in the thoracic ganglion, and their relative positions were determined. The somata of MN1-4 lie in a cluster located near the lateral surface of the thoracic ganglion at the border of the pro- and mesothoracic regions, ipsilateral to the muscle fibers innervated. The somata of MN1 and 2 lie side by side in a horizontal plane with MN1 in a more anterior position. Those of MN3 and 4 lie ventrally to those of MN1 and 2 in a horizontal plane with MN3 in the more anterior position. The soma of MN5 is located contralaterally to the muscle fiber it innervates, lying in the dorsal outermost layer of the thoracic ganglion next to the midline at the border of the pro- and mesothoracic regions.(ABSTRACT TRUNCATED AT 250 WORDS)