In this work, making use of experimental designs, headspace-stir bar sorptive extraction (HS-SBSE) followed by thermal desorption (TD) coupled to a gas chromatography-mass spectrometry (GC-MS) for the simultaneous determination of mercury and tin organometallic compounds present in surface water, sediment and biological tissue was optimized. All solid samples require a previous extraction typically done with diluted HCl or KOH/methanol solutions, and the derivatization, in all the cases, of the organometallic compounds with NaBEt(4). As a consequence, the preconcentration step was carried out in a 0.1 mol L(-1) HOAc/NaOAc buffer solution, with 0.1% (m/v) of NaBEt(4), without the addition of NaCl as a salting out reagent, and with the stir bar (20 mm x 1 mm) located in the headspace (HS). In addition, the desorption step required the following conditions: 250 degrees C (desorption temperature), 15 min (desorption time), 14.1 psi (approximately 97.2 kPa) (vent pressure) and 75 mL min(-1) (vent flow). Finally, to assure the extraction of all the analytes under equilibrium, 5h are required. Inorganic mercury (Hg(2+)) and tripropyltin (TPrT) were used as internal standards to correct for variations in the extraction, derivatization and detection steps. The resulting method provides precise (4-17%) and accurate (against four certified reference materials) results in the ng L(-1) and pg g(-1) range concentrations with recoveries within 80-120% for water samples. The proposed methodology is currently applied in the speciation analysis of specimens obtained in different estuarine sites of the Basque Coast.