Novel N,N-disubstituted indol-3-ylglyoxylamides (1-56), bearing different combinations of substituents R 1-R 5, were synthesized and evaluated as ligands of the translocator protein (TSPO), the 18 kDa protein representing the minimal functional unit of the "peripheral-type benzodiazepine receptor" (PBR). Most of the new compounds showed a nanomolar/subnanomolar affinity for TSPO and stimulated steroid biosynthesis in rat C6 glioma cells with a potency similar to or higher than that of classic TSPO ligands such as PK 11195. Moreover, when evaluated in vivo by means of the elevated-plus-maze (EPM) paradigm in the rat, compound 32, the best-performing derivative in terms of TSPO affinity and pregnenolone production, showed clear anxiolytic effects. The results of this study suggested that the novel N,N-disubstituted indol-3-ylglyoxylamides may represent a promising class of compounds potentially suited for the treatment of anxiety disorders.