A quantitative screening for intra- and extracellular lipolytic activity was performed in submerged cultures of four Thermus strains using two different media (named T or D medium). Major differences in the extracellular lipolytic activity were observed in T medium, the highest values being for Thermus thermophilus HB27 and Thermus aquaticus YT1 strains (18 and 33 U/L, respectively). Two enzymes with lipase/esterase activity were identified in the four Thermus strains by zymogram analysis, with molecular weights of 34 and 62 kDa. No kinetic typification of the enzymes as primary metabolites was possible for any of the Thermus strains, because of the lack of a good fitting of the experimental lipolytic activity production rates to the Luedecking and Piret model. However, a linear relationship was found between the absolute values of biomass and total lipase/esterase activity (sum of intracellular and extracellular). For T. thermophilus HB27, an increase in the aeration rate caused the increase in the production of biomass and, particularly, intracellular lipolytic activity but the extracellular lipolytic activity was not affected except for the series with the strongest oxygen limitation. Transmission electronic microscopy revealed that T. thermophilus HB27 formed rotund bodies surrounded by a common membrane in cultures in the early stationary phase. The results suggest the occurrence of a specific mechanism of lipase/esterase secretion that might be due to the different composition and permeability of the cell membranes and those surrounding the rotund bodies.