High-affinity, specific 3H-5-hydroxytryptamine (5-HT) binding was analyzed in membrane homogenates of human frontal cortex, caudate, and globus pallidus. 5-HT1A and 5-HT1C binding sites were pharmacologically blocked using 100 nM 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) and 100 nM mesulergine, respectively. The majority of 5-HT1 sites remained in each of the three brain regions under these conditions. The pattern of nucleotide interactions with these binding sites (GppNHp = GTP = GDP greater than GMP = adenine nucleotides) suggests a possible linkage to a G protein. RU 24969 competition studies confirmed the absence of 5-HT1B binding sites in human cortex, caudate, and globus pallidus. Drug interactions with putative 5-HT1D binding sites in bovine caudate membranes correlated significantly with their affinities for human membrane recognition sites labeled by 3H-5-HT in the presence of 100 nM 8-OH-DPAT + 100 nM mesulergine. We conclude that the majority of 3H-5-HT labeled recognition sites in human cortex, caudate, and globus pallidus represent 5-HT1D binding sites.