In this study, several fermentation media were tested for the production of penicillin G acylase (PCA) using Bacillus megaterium. The carbon sources studied were glucose and lactose. The nitrogen sources studied were enzymatic casein hydrolysates produced with proteases of different specificities. The replacement of glucose with cheese whey and the addition of free amino acids in the PGA production were also tested. The results showed a strong correlation between the nitrogen source and enzyme yield and the presence of glucose repression. The highest enzyme concentration achieved was 138 IU/L using casein hydrolyzed with 0.6 L of Alcalase(R) and cheese whey.