5-hydroxytryptamine (5-HT) is known to modulate the locomotion generator network in the lamprey spinal cord, but little is known about the pattern of 5-HT innervation along the spinal cord. The distribution of 5-HT-immunoreactive (5-HT-ir) cells and fibers, as well as the effects of 5-HT on the locomotor network in the rostral and caudal parts of the spinal cord were compared in two lamprey species, Lampetra fluviatilis and Petromyzon marinus. Intraspinal 5-HT cells form a very dense ventromedial plexus in which the dendrites of neurons forming the locomotor network are distributed. The number of 5-HT cells and varicosities in this plexus decreases in the fin area (segments 70-90), and then increases somewhat in the most caudal segments. The descending 5-HT fibers from the rhombencephalon are located in the lateral and ventral columns, and their numbers gradually decrease to around 50% in the tail part of the spinal cord. In contrast, the number of 5-HT-ir axons in the dorsal column remains the same along the spinal cord. Bath application of both N-methyl-D-aspartic acid (NMDA, 20-250 microM) and D-glutamate (250-1000 microM) was used to induce fictive locomotion in the isolated spinal cord. Bath application of 5-HT (1 microM) reduced the burst frequency in the presence of NMDA. The 5-HT effect was, however, significantly greater in the rostral as compared to the caudal part. With D-glutamate, the 5-HT effects was instead more pronounced in the caudal spinal cord. To account for this difference in 5-HT effects on NMDA- and D-glutamate-induced fictive locomotion, the cellular effect of D-glutamate was further investigated. It activates not only NMDA, but also alpha amino-3-hydroxy-5-methyl-4-isoxyl propionate (AMPA)/kainate and metabotropic glutamate receptors. In contrast to NMDA, D-glutamate did not elicit tetrodotoxin (TTX)-resistant membrane potential oscillations. This difference in action between NMDA (selective NMDA receptor agonist) and D-glutamate (mixed agonist) may partially account for the differences in effect of 5-HT on the locomotor pattern.