We compared two methods for the preservation of fungi, Castellani's method and repeated passage in Sabouraud medium-agar, on five isolates of Sporothrix schenckii that were preserved for 18 years at room temperature by both procedures. They were evaluated for viability of the strains, growth rate, morphological and physiological characteristics, and in vitro sensitivity to iodide, itraconazole, terbinafine and posaconazole. 100% viability was observed in all of the isolates, with slower growth rate on strains preserved in water compared to strains periodically re-cultured. The typical morphological feature of these fungi was preserved by both methodologies. With regard to enzymatic activity, both groups gave urease reactions and were beta glucosidase-positive. Nevertheless, complete inhibition of the capacity to hydrolyse starch was observed only on the isolates preserved in water. This group also was more sensitive to potassium iodide at a concentration of 10 microM in the in vitro sensitivity tests.