This study was undertaken to investigate the pharmacology of human serotonin (5-HT)1D receptor sites by measuring two functional cellular responses, inhibition of forskolin-stimulated cAMP formation and promotion of cell growth, using transfected rat C6-glial cell lines and a broad series of 5-HT receptor agonists. Stable and separate transfection of a pcDNA3 or pRcRSV plasmid, each containing a cloned human 5-HT1D receptor gene, in rat C6-glial cells was confirmed with RT-PCR of 5-HT1D receptor mRNA and radioligand binding with [3H] 5-carboxamidotryptamine (5-CT) and [3H] sumatriptan. The 5-HT1D receptor density was 350 and 1050 fmol/mg protein for the C6-glial/pcDNA3/5-HT1D and C6-glial/pRcRSV/5-HT1D cell line, and forskolin (100 microM)-induced cAMP formation was inhibited by 45 and 78% in the presence of 1 microM 5-HT, respectively. A comparison of the intrinsic agonist activities for sixteen 5-HT receptor ligands with their corresponding binding affinities for the human 5-HT1D receptor site showed similar results for both cell lines with the exception of the partial agonist m-trifluoro-phenyl-piperazine (TFMPP). Three classes of compounds were observed: 1) efficacious agonists, such as 5-CT, 5-methoxytryptamine, 5-HT, sumatriptan, bufotenine, 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)1H-indole (RU 24,969), tryptamine and 8-hydroxy-2(di-n-propilamino)tetralin (8-OH-DPAT), with agonist potency close to their binding affinity; 2) the partial agonists metergoline, 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrolo-(1,2-a) quinoxaline (CGS 12066B), 1-naphthylpiperazine and 2'-methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-amide (GR 127,935) with marked intrinsic agonist activity but at concentrations higher than their binding affinity; and 3) the silent antagonists ritanserin, ketanserin and methiothepin, apparently free of intrinsic agonist activity, with antagonist potency close to their binding affinity. The cAMP data were further supported by the observed promotion of cell growth by stimulation of both transfected cell lines with sumatriptan under serum-free conditions; half-maximal stimulation was obtained at 4.4 nM (C6-glial/pcDNA3/5-HT1D) fully in agreement with its EC50-value (5.7 nM) for inhibition of cAMP formation. This growth promoting effect was antagonised by 1 microM methiothepin and not observed in pcDNA3-plasmid-transfected and non-transfected C6-glial cells. A comparative study with a C6-glial/pcDNA3/5-HT1B cell line expressing a similar amount of cloned human 5-HT1B receptors (Bmax: 360 fmol/mg protein) showed almost no intrinsic agonist activity for metergoline, 1-naphtylpiperazine and GR 127,935. Together with the 5-HT1D receptor binding selectivity and antagonist activity of ketanserin and ritanserin, the findings define important pharmacological differences between cloned human 5-HT1D and 5-HT1B receptor sites.