Abstract The aim of this study was to assess protease production and virulence of various Burkholderia pseudomallei strains. Protease activity was evaluated in filtrates from cultures grown for 50 h in TSB Dialysate by azocasein hydrolysis, and expressed as absorbancy at 405 nm. Virulence was assessed in 8 weeks old SWISS mice, by intraperitoneal injection of 6–6×10 5 CFU, and the LD 50 was calculated after 30 days by the method of Reed and Muench. The lethal activity was studied for five strains of B. pseudomallei and the type strains of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia cepacia. The three type strains appeared to be low protease producers ( A 405=0.11, 0.09 and 0.00, respectively) and avirulent. The two more virulent B. pseudomallei strains exhibited significantly different LD 50, 3.5×10 2 (IPP 6068 VIR) versus 2.1×10 5 CFU/mouse (40/97), and protease activities ( A 405=0.046 and 0.79, respectively). Moreover, the avirulent parent of IPP 6068 (AG), was a better protease producer than the 6068 VIR strain, A 405=0.26 versus 0.046. These results suggest that there is no correlation between virulence and level of exoproteolytic activity, when B. pseudomallei is injected to mice via the intraperitoneal route.