<P><B>Abstract</B><P>The high cost of cellulolytic enzymes used in the ethanol production process has led to a growing interest in situ production. The evaluation of the influence of several factors in the fungus Penicillium sp. cellulase production using pretreated sugarcane bagasse is very interesting. Penicillium sp. cellulase production by using filter paper as cellulosic substrate and the use of glucose, sucrose and lactose like co-substrates was assessed. In the experiments using filter paper as a cellulosic substrate, the highest FPase enzyme activity obtained was 280 FPU.L-1 using sucrose as co-substrate. Subsequently, the study of pretreated sugarcane bagasse was conducted using Plackett-Burman experimental design with analysis of 6 factors influencing the process. The highest FPase activity was 615.1 FPU.L-1. The factors influencing FPase and β- glucosidase activity were the use of molasses and the solid loading. The successful use of molasses as co-substrate opens perspectives for future researches.</P></P>