In vitro and ex vivo measurements have shown that the binding of the selective high-affinity agonist, S14506 (1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxy-naphthyl)piperazine), to 5-HT1A receptors, is similar in affinity (Kd = 0.79 nM) and extent (Bmax) to that of the antagonist, WAY 100635. We aimed to test whether S14506, labeled with a positron- emitter, might serve as a radioligand for imaging brain 5-HT1A receptors in vivo with positron emission tomography (PET). Here we evaluated [11C]S14506 and [18F]S14506 in rat and rhesus monkey in vivo. After intravenous administration of [11C]S14506 into rat, radioactivity entered brain, reaching 210% SUV at 2 min. Radioactivity uptake into brain was higher (∼ 350% SUV) in rats pre-treated with the P-glycoprotein (P-gp) inhibitor, cyclosporin A. In rhesus monkey, peak brain uptake of radioactivity after administration of [11C]S14506 or [18F]S14506 was also moderate and for [11C]S14506 increased from ∼ 170% SUV after 7 min, to 240% SUV in a monkey pre-treated with the P-gp inhibitor, tariquidar. The ratios of radioactivity in 5-HT1A receptor-rich regions, such as cingulate or hippocampus to that in receptor- poor cerebellum reached between 1.35 and 1.5 at 60 min for both [11C]S14506 and [18F]S14506. [11C]S14506 gave one major polar radiometabolite in monkey plasma, and [18F]S14506 gave three and two more polar radiometabolites in rat and monkey plasma, respectively. The rat radiometabolites of [18F]S14506 did not accumulate in brain. [18F]S14506 was not radiodefluorinated in monkey. Thus, despite high-affinity and lack of troublesome brain radiometabolites, both [11C]S14506 and [18F]S14506 were ineffective for imaging rat or monkey brain 5-HT1A receptors in vivo, even under P-gp inhibited conditions. Explanations for the failure of these radioligands are offered.