Centesimal dilutions (5, 9 and 15 cH) of Gelsemium sempervirens are claimed to be capable of exerting anxiolytic and analgesic effects. However, basic results supporting this assertion are rare, and the mechanism of action of G. sempervirens is completely unknown. To clarify the point, we performed a comparative analysis of the effects of dilutions 5, 9 and 15 cH of G. sempervirens or gelsemine (the major active principle of G. sempervirens) on allopregnanolone (3α,5α-THP) production in the rat limbic system (hippocampus and amygdala or H-A) and spinal cord (SC). Indeed, H-A and SC are two pivotal structures controlling, respectively, anxiety and pain that are also modulated by the neurosteroid 3α,5α-THP. At the dilution 5 cH, both G. sempervirens and gelsemine stimulated [(3)H]progesterone conversion into [(3)H]3α,5α-THP by H-A and SC slices, and the stimulatory effect was fully (100%) reproducible in all assays. The dilution 9 cH of G. sempervirens or gelsemine also stimulated 3α,5α-THP formation in H-A and SC but the reproducibility rate decreased to 75%. At 15 cH of G. sempervirens or gelsemine, no effect was observed on 3α,5α-THP neosynthesis in H-A and SC slices. The stimulatory action of G. sempervirens and gelsemine (5 cH) on 3α,5α-THP production was blocked by strychnine, the selective antagonist of glycine receptors. Altogether, these results, which constitute the first basic demonstration of cellular effects of G. sempervirens, also offer interesting possibilities for the improvement of G. sempervirens-based therapeutic strategies.