The paper describes a novel extraction procedure for lipopolysaccharides (LPS) from Salmonella enterica subsp. enterica (PCM 2266). Process parameters for the extraction of LPS from bacterial mass were optimized by carrying out a two-level fractional design experiment. Four parameters, namely temperature, CO2 flow rate, pressure and co-solvent composition were analyzed. The best crude extract yields were achieved when the CO2 flow rate and temperature were kept high (10 g/min, 90 degrees C) and pure water was used as a co-solvent. Pressure had no statistically significant effect within the range of the study performed, whereas the other factors were relevant. The recovery of the extracted LPS by scCO(2) was about 3.3% of the biomass used, while in the classical extraction method yield was less than 2%. All isolates were characterized by SDS-PAGE, by the spectra of the thiobarbituric acid reaction products and GLC-MS analysis. (C) 2007 Elsevier B.V. All rights reserved.