BACKGROUND: The use of agroindustrial residues as substrate for inulinase bio-production is a good choice to reduce production costs, since enzyme activity will be improved and the downstream step of the process will be viable technically and economically. In addition, the screening of microorganisms that are able to overproduce inulinase using these substrates is fundamental to guarantee successful medium substitution. Based on these considerations, the objective of this present work was to select different strains of yeasts of the genus Kluyveromyces to produce the inulinase. RESULTS: Initially, 10 strains were tested in synthetic medium and six of these selected to perform tests in agroindustrial medium. Screening showed that the strains NCYC 587 and NRRL Y-7571 were able to produce the enzyme using agroindustrial residues as substrate. Optimization of inulinase activity as a function of pH, and concentrations of molasses, corn steep liquor (CSL) and yeast extract (YE) was carried out for the two strains. The maximum inulinase activity under optimized conditions was about 750 U mL(-1). CONCLUSION: These values are approximately seven times greater than the values obtained using synthetic medium, showing the technical viability of the use agroindustrial residues for the inulinase production. (C) 2009 Society of Chemical Industry