Kinetics of cell growth and protease production by four proteolytic bacterial strains, namely, Bacillus subtilis EMCC 1020, Bacillus megaterium EMCC 1057, Serratia marcescens EMCC 1247, and Pseudomonas fluorescens EMCC 1221, in milk permeate, compared with other fermentation media, were studied. The pH values, lactose utilization, and biochemical oxygen demand (BOD) reduction in milk permeate also were investigated. The four strains were able to grow in milk permeate, and to produce considerable amounts of protease, reaching 289 (EMCC 1020), 252 (EMCC 1057), 263 (EMCC 1247), and 212 (EMCC 1221) U/mL after 30 h of fermentation. The growth and enzyme activity of the four strains were greater in milk permeate than those in other fermentation media. The protease-producing bacteria were able to utilize lactose in milk permeate with values ranging from 37.62 to 54.97% and to reduce the BOD of milk permeate by 50.59-63.65%. Milk permeate proved to be the best medium for enzyme production by all organisms examined.