Benzodiazepines induce an immediate anxiolytic activity at the expense of side effects such as sedation, tolerance and withdrawal. In contrast, selective serotonin receptor uptake inhibitors (SSRIs) are known to offer long-term symptom improvement without inducing tolerance and withdrawal, but with a delayed onset of the anxiolytic effect. ELB139 is a novel agonist at the benzodiazepine binding site with pronounced anxiolytic and anticonvulsant activity without inducing tolerance to both effects after chronic administration. ELB139 shows a selectivity for alpha-3-subunit containing GABA(A) receptors. In the present study the effect of the compound on monoaminergic neurotransmitter levels were investigated by microdialysis. ELB139 induced a significant increase of extracellular 5-HT in the striatum and the medial prefrontal cortex of rats without affecting dopamine levels in these areas. The increase of 5-HT in the striatum was reversed by systemic and by local administration of the benzodiazepine antagonist flumazenil in the dorsal raphe nucleus by a microdialysis probe, suggesting that the increase in 5-HT was mediated by the activity of ELB139 at the benzodiazepine binding site. As the dorsal raphe nucleus is rich in alpha-3 subunits, this effect of ELB139 may be mediated by its subtype selectivity. Thus, ELB139 seems to combine effects seen with benzodiazepine agonists and SSRIs in one compound.