Enzymes stable and active in non-aqueous media are in large demand for their increasing application in organic synthesis. A solvent-stable protease is reported from a solvent tolerant strain (PseA) of Pseudomonas aeruginosa. Production of this novel protease was optimized by varying the culture conditions. Glycerol (0.7%) as carbon source, casein (0.4%) and yeast extract (0.6%) as nitrogen sources, CaCl2 (0.6 mM) as metal ion in culture media (pH 7.0), inoculated with 2.5% of 36 h grown mother culture and incubation for 24 h at 30 degrees C with 250 rpm shaking were found to be the best culture conditions leading to maximum growth and protease production (1601 U/ml). The enzymatic properties of the protease revealed, pH and temperature optimum of 9.0 and 55 degrees C, respectively. It retained 100%, 91% and 80% of its initial activity after heating for 30 min at 45, 50 and 55 degrees C, respectively. The enzyme exhibited remarkable stability towards range of hydrophobic organic solvents. This unique property makes it attractive for use in non-aqueous enzymology. (C) 2007 Elsevier Inc. All rights reserved.