High affinity, specific [(3)H]5-hydroxytryptamine (5-HT) binding to spinal cord synaptosomes was examined to identify the 5-HT receptor subtypes present. Computer nonlinear regression analysis of competition studies employing 8-OH-DPAT indicated that this 5-HT(1A) selective agonist demonstrated high affinity competition (K(i = 1.3 nM)) for 24.6 +/- 0.7% of the total [(3)H]5-HT binding sites. Competition studies employing the 5-HT(1B) selective agonist RU24969, in the presence of 100 nM 8-OH-DPAT, indicated that RU24969 demonstrated high affinity (K(i = 1.1 nM)) competitive inhibition for 26.2 +/- 1.4% of all [(3)H]5-HT binding sites. Neither 5-HT(1C), 5-HT(1D), 5-HT(2) nor 5-HT(3) selective compounds demonstrated any high affinity competition for the residual 49% of specific [(3)H]5-HT binding. Therefore, three major classes of [(3)H]5-HT binding sites could be demonstrated in spinal cord synaptosomes: 5-HT(1A), 5-HT(1B) and a novel [(3)H]5-HT binding site which respectively represented 25, 26 and 49% of spinal cord synaptosomal [(3)H]5-HT binding. Further studies focusing on the function of the latter binding site are needed to determine if the presently identified novel binding site is the major 5-HT(1) receptor subtype present in spinal cord.