Bioavailability of the pharmaceutical substances is very important for their activity. In case of necessity, bioavailability can be improved by reducing the particle size of the drugs. In this study, particle size of digitoxin was reduced by the Rapid Expansion of Supercritical Solutions (RESS). The effects of pre-expansion temperature (90-110 degrees C), flow rate (2.5-7.5 ml/min), spray distance (3-7 cm) on the size and size distribution of the precipitated digitoxin particles were carried out. The particles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and LC-MS analysis. While the particle size range of the original digitoxin was 0.2-8 mu m, it was decreased to 68-458 nm and determined that 97% of the particles were below 200 nm depending on the different experimental conditions. Response surface method (RSM) was used to optimize the process parameters. The flow rate, 7 ml/min; spray distance, 7 cm; pre-expansion temperature, 95 degrees C were found to be the optimum conditions to achieve the minimum particle size of digitoxin. (C) 2009 Elsevier B.V. All rights reserved.