We have stabilized alcalaseTM and subtilisin Carlsberg (SC) against heat by chemical modification with ethylene glycol bis-succinimidyl succinate (EGNHS), a procedure not previously reported for subtilisins. The increases in thermal stability at 65oC were 1.8-fold and 4.7-fold respectively. Caseinolytic activity of alcalase in aqueous buffer was unchanged following modification but apparent Km of SC decreased 2.5-fold. Native and modified forms of both enzymes synthesized the tripeptide Z-Tyr-Gly-Gly-NH2 under kinetic control in mixtures based on 0.2M barbitone buffer, pH 9.0 and 50% v/v dimethyl formamide/ barbitone buffer. Native enzymes gave faster rates of product formation than their modified counterparts in buffer but differences were much less pronounced in the mixed solvent. We also compared native alcalase and SC in terms of thermal stability, tolerance of organic solvents and autolysis. Alcalase was approx. 4.6-fold more stable than SC at 65oC and was more tolerant of acetone, acetonitrile and 1,4-dioxane. Alcalase underwent autolysis at approx. half the rate of SC. Against succinyl- Ala-Ala-Pro-Phe substrates, alcalase showed a much higher esterase/ amidase ratio (567) than SC (29) in aqueous buffer but this was reversed in 50% v/v dimethylformamide, where the esterase/ amidase ratios were 43 and 113 respectively.