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 > 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.