Two microorganisms, Bacillus subtilis and Serratia marcescens, have been selected in order to assess their ability to produce proteases. First, their performances have been studied using three media providing different levels of the main nutrients (complex, semi-defined and defined). The optimal absolute production was obtained in the complex media, while maximum protease activity per cell weight appeared in defined medium for Bacillus and in semi-defined and complex media for Serratia. Then, the effects of applying different environmental conditions to the cells were studied. The two microorganisms were immobilised in calcium alginate beads, protease production by these systems was assessed in the previously tested culture media, and the performances in free and immobilized operating conditions were compared. The richest medium seemed to be the best one in terms of absolute protease production, although the use of semi-defined or defined media could be considered more appropriate, in order to minimise growth of cells leaked from the support, and therefore downstream processing cost. Productivity was slightly higher in free cultures than in immobilized cultures, and retention of enzyme within the alginate beads was detected in the latter, indicating the occurrence of diffusional Limitations. In all the cases studied, Serratia marcescens appeared as a better protease producer than B. subtilis, in terms of absolute production. This fact could be related to the different cell growth levels observed for the two microorganisms.