A new method involving headspace single-drop microextraction (SDME) with in-drop derivatization and CE is developed for the preconcentration and determination of free cyanide. An aqueous microdrop (5 mu L) containing Ni(II)-NH3 (as derivatization agent), sodium carbonate and ammonium pyromellitate (as internal standard) was used as the acceptor phase. The extracted cyanide forms a stable Ni(CN)(4)(2-) complex which is then determined by CE. Common experimental parameters (sample and acceptor phase pH, extraction temperature, extraction time and sample ionic strength) affecting the extraction efficiency were investigated. Using headspace SDME, free cyanide can be effectively extracted from the neutral solutions, i.e. without the acidification of the sample which often is prone to errors due to incomplete liberation and artefactual cyanide production. Proposed SDME-CE method provided about 58-fold enrichment in 20 min. The calibration curve was linear for concentrations of CN- in the range from 0.25 to 20 mu mol/L (R-2 = 0.997). The LOD (S/N = 3) was estimated to be 0.08 mu mol/L of CN-. Such a detection sensitivity is high enough for free cyanide determination in common environmental and physiological samples. Finally, headspace SDME was applied to determine free cyanide in human saliva and urine samples with spiked recoveries in the range of 91.7-105.6%. The main advantage of this method is that sample clean-up, preconcentration and derivatization procedures can be completed in a single step. In addition, the proposed technique does not require any sample pretreatment and thus is much less susceptible to interferences compared to existing methods.