Nowadays, there is not much information on the large-scale production of thermostable lipolytic enzymes by thermophilic organisms. Therefore, in this study the lipolytic enzyme production by Thermus thermophilus HB27 in laboratory-scale bioreactors was undertaken. In order to determine the most suitable bioreactor, two configurations were investigated: stirred-tank and airlift bioreactor. It was demonstrated that the stirred-tank configuration led to the highest lipolytic extracellular activities, about 2.3-fold higher than those found in the corresponding cultivation in the airlift bioreactor. On the other hand, the influence of several factors such as culture nutrients concentration, aeration, and agitation rate on the production of thermophilic lipolytic enzymes in a 5-L stirred-tank bioreactor was assayed. At reduced nutrients concentration (50 % with respect to the basal medium), a higher product/biomass yield was attained, without any operational problems. From the relationship between mass transfer coefficient (K(L)a), aeration, and agitation rates it was concluded that there is a lesser dependence on the aeration than the agitation rate. In addition, the mechanical stirring of the bioreactor could tear the membranes that contain the rotund bodies often found in cultures of thermophilic microorganisms, thus increasing the extracellular enzyme production.