One of the challenging questions in benzodiazepine (BDZ) research is the identification of endogenous BDZ ligands in the brain, which could modulate gamma-aminobutyric acid neurotrasmission. It has been established that endogenous substances with benzodiazepine-like activity play a role in the pathogenesis of different pathologies like hepatic encephalopathy or idiopathic recurring stupor , several substances with benzodiazepine-like activity were also found in food and in several officinal plants used in folk medicine as hypnotic or tranquillisers such as and matricaria chamomilla. The presence of molecules with high affinity for central and peripheral benzodiazepine receptors was also determined in the pod and leaves of ceratonia siliqua. The aim of the present work was to determine the possible presence of BDZ-like compounds in leaves of nicotiana tabacum and to test their ability to bind central or peripheral BDZ receptors. Tobacco leaves have been extracted with methanol and the crude extract was chromatographed at 0.8 ml/min on a lichrospher 100 RP-18 column (250x4.0 mm; 5 micrometer) equilibrated with 80% water/1% FA and 20% acetonitrile. The sample was analysed using a water/1%fa and acetonitrile gradient at 0.5% from 20 to 58% acetonitrile. 75 fraction were collected, lyophilised and tested for their ability to inhibit [3h]RO 15-1788 specific binding to central BDZ binding site or [3h]PK 11195 specific binding to peripheral BDZ binding site (PBR). The crude extract, as well as the separated HPLC fractions, have been analyzed by HPLC-MS and HPLC-MS-MS in order to identify any substances related to the observed biological activity. Collision induced decomposition (CID) spectra allowed to identify some possible compounds, such as steroids and glucosides of different nature. The analysis of the separated fractions showed that these components are spread along several fractions, suggesting a quite complex mixture and high concentration, which tend to affect the chromatographic behaviour of the purification step. Future work will focus on MS-MS scan analysis directed at families of compounds and will combine HPLC-MS-MS to biological activity data in order to identify ions strictly related to the activity.