The localization of calbindin-D28K (CB) was studied immunocytochemically in laminae I and II of the dorsal horn and in spinal ganglia in the chicken, and compared with the distribution of substance P (SP) using double immunolabeling. At the light microscopic level, CB immunoreactivity was observed most intensely in the lamina II using the avidin-biotinylated peroxidase complex (ABC) and immunofluorescence methods. At the electron microscopic level using the ABC method, CB immunoreactivity was observed in the following three neuronal elements: 1) the scalloped central terminal with many dense-cored vesicles (DCVs) in the synaptic glomerulus; 2) some vesicle-containing dendrites (VCDs) inside or outside the synaptic glomerulus; and 3) some axon terminals outside the synaptic glomerulus. The CB-immunoreactive (IR) VCDs in the synaptic glomerulus often formed reciprocal synapses with the central terminal. Strong immunoreactivity was observed at the postsynaptic membrane of CB-IR elements. Double immunofluorescence and immunolabeling methods at the electron microscopic level showed that CB and SP colocalized in the scalloped central terminal with DCVs of the synaptic glomerulus. Almost all SP-IR neurons in the spinal ganglion revealed the coexistence of CB in serial sections in the chicken. In light of previous biochemical and physiological reports, our findings suggest that CB - coexisting with SP - plays an important role in the control of pain transmission through its strong Ca(2+)-buffering action in the chicken.