High-intensity pulsed electric field (HIPEF) is a nonthermal food processing technology that is currently being investigated to inactivate microorganisms and certain enzymes, involving a limited increase of food temperature. Promising results have been obtained on the inactivation of microbial enzymes in milk when suspended in simulated milk ultrafiltrate. The aim of this study was to evaluate the effectiveness of continuous HIPEF equipment on inactivating a protease from Bacillus subtilis inoculated in milk. Samples were subjected to HIPEF treatments of up to 866 mus of squared wave pulses at field strengths from 19.7 to 35.5 kV/cm., using a treatment chamber that consisted of eight colinear chambers connected in series. Moreover, the effects of different parameters. such as pulse width (4 and 7 mus), pulse repetition rates (67, 89, and 111 Hz), and milk composition (skim and whole milk) were tested. Protease activity decreased with increased treatment time or field strength and pulse repetition rate. Regarding pulse width, no differences were observed between 4 and 7 mus pulses when total treatment time was considered. On the other hand, it was observed that milk composition affected the results since higher inactivation levels were reached in skim than in whole milk. The maximum inactivation (81%) was attained in skim milk after an 866-mus treatment at 35.5 kV/cm and 111 Hz.