Toxins produced by Bacillus thuringiensis require proteolytic processing to become active. The activation of B. thuringiensis subsp. medellin Cyt1Ab1 cytolytic toxin by trypsin, chymotrypsin and gut extracts from Culex quinquefasciatus was analyzed. The toxin was processed into fragments between 25 and 26 kDa. An effect of the pH on toxin proteolysis was observed, the highest activity was achieved at the pH value of 12. The in vivo proteolytic processing by third instar C. quinquefasciatus larvae and the in vitro processing with their gut extracts showed similar results. The solubilized toxin and protease resistant cores generated by in vitro processing showed hemolytic activity but they did not show mosquitocidal activity in vivo. The results support the role of proteases in the activation of B. thuringiensis subsp. medellin Cyt1Ab1 toxin.