The terminal projections of the descending 5-hydroxytryptamine (5-HT) bulbospinal pathway and the coexistence among 5-HT-, substance P (SP)-, and thyrotropin-releasing hormone (TRH)-like immunoreactivities (LI) in fibers innervating the L7 segment in the cat spinal cord were studied quantitatively and semiquantitatively by use of the indirect double-staining immunofluorescence technique. The content of 5-HT, SP, and TRH in different parts of the spinal cord was determined by use of radioimmunoassay (RIA) (SP and TRH) and high-performance liquid chromatography with electrochemical detection (HPLC-ECD) (5-HT). For all three substances studied, immunoreactive (IR) axon terminals were found in all parts of the gray matter, but with clear regional variation in the density of innervation. Thus, all three substances showed a dense innervation in the motor nucleus, particularly in the ventral part of the nucleus, while the superficial dorsal horn was very densely innervated by SP-IR fibers (laminae I and II) and TRH-IR fibers (laminae II and III). In the motor nucleus, the studied substances coexisted to a very high degree, but some 5-HT-IR fibers (about 10%) lacked peptide-LI and some SP-IR fibers (about 10%) lacked 5-HT-LI while virtually all TRH-IR fibers also contained 5-HT-LI. In the superficial dorsal horn (laminae I-III), no coexistence was detected, while other parts of the gray matter displayed various degrees of coexistence in between those found in the motor nucleus and laminae I-III. The quantitative analysis of IR varicosities in the motor nucleus suggested that the unilateral L7 motor nucleus is innervated by about 55-110 x 10(6) 5-HT-IR nerve terminals, which may indicate as many as 4,000 boutons per descending 5-HT cell body in the brain stem only with this restricted projection. When combing these results with the biochemical data, it could be calculated that the concentration of 5-HT in IR varicosities is about 3-6 x 10(-3) M, while the corresponding figures for SP and TRH was 0.3-0.5 x 10(-3) M and 0.1-0.2 x 10(-3) M, respectively. In cats subjected to spinal cord transection at the lower thoracic level, all 5-HT-IR fibers in the L7 segment had disappeared 44 days after the lesion, indicating a strict suprasegmental origin of 5-HT-IR fibers in this segment.(ABSTRACT TRUNCATED AT 400 WORDS)