Ascosphaera apis (Maassen ex Claussen) Olive and Spiltoir, is the causative agent of chalkbrood disease in honey bees (Apis mellifera L.). The process of infecting honeybee colonies is variable and depends on many agents: the propriety of the pathogen, behavior resistance in the honey bee colony, the genetic potential of the queen, environmental conditions, etc. The aim of the present study was to determine the enzymatic activity of 45 strains of Ascosphaera apis isolated from apiaries from Poland using the API ZYM system. All strains of A.apis were cultured for 8 days on SDA-YE with an additional 0.1% chloramphenicol, maintained at 25degreesC. Two to three portions of mycelium were taken from each culture, placed in separate sterile grinders with 1-2 ml of suspension medium and homogenized until a uniform turbid mycelial suspension was obtained. The suspension was standardized to McFarland No. 4 with suspension medium. Samples were tested utilizing API ZYM test strips (BioMerieux) according to the manufacturer's procedure. None of the 45 strains A.apis produced either lipase C 14, trypsin, alpha-galactosidase, beta-glucuronidase or alpha-fucosidase. Enzymatic profiles from 45 strains of A. apis demonstrate that there is a high specialization of this mycotic agent in relation to the brood of honeybee. The results confirm the genus attachment of the strains that do not produce characteristic fructification. Enzymatic profiles are a very useful tool in laboratory diagnostic work.