It is the first time when psychrotrophic, solvent tolerant, psychro-thermoalkaline protease producing strain of Pseudomonas putida isolated from dairy sludge was capable of growing in the presence of 30% (v/v) organic solvents. The strain exhibited resistance against heavy metals (Cr(6+), As(3+), Pb(2+), Cs(1+)) and various antibiotics. The isolate was able to grow at wide range of temperature (10-40 degrees C) with maximum growth at 25 degrees C. In glucose gelatin yeast extract (GGY) broth (pH 9.0 and 25 degrees C), the strain produced 514 U Protease ml(-1). The presence of organic solvents n-dodecane, n-decane, isooctane and n-octane, enhanced the protease production. The protease was not only stable but also its activity enhanced in the presence of 25% (v/v) solvents n-dodecane, n-decane, isooctane, n-octane, n-hexane, n-butanol, n-heptane, cyclohexane and xylene, after prolonged incubation of fourteen days. The molecular weight of purified protease was similar to 53 kDa as revealed by SOS-PAGE and activity gel analysis. The protease was active in broad pH and temperature range of 8.0-12.0 and 10-70 degrees C, respectively, with maxima at pH 9.5 and 40 degrees C. The unique property of solvent tolerance, antibiotic and heavy metal resistance proves the potential candidature of this isolate not only for peptide synthesis, but also for industrial use and bioremediation strategies involved in environmental clean up. (C) 2011 Elsevier Ltd. All rights reserved.