The species specificity of the Candida krusei DNA fingerprinting probe CkF1,2 has been investigated. A total of 149 pathogenic and nonpathogenic fungal and bacterial DNAs were screened with CkF1,2. The probe was cold labeled with peroxidase, and its specificity was assessed by using Southern blot, dot blot, and colony blot hybridization. Its sensitivity was determined by dot blot hybridization. The CkF1,2 probe proved to be species specific. It hybridized with DNA for the 112 C. krusei strains studied, whereas it failed to hybridize under low-stringency conditions to 37 DNAs from 27 different yeast species, including Candida albicans, Candida glabrata, Candida norvegensis, Candida inconspicua, Candida tropicalis, Candida valida, Candida zeylanoides, and Yarrowia lipolytica, as well as DNAs from the filamentous fungi and bacteria tested. However, CkF1,2 hybridized strongly with DNA of the yeast species Issatchenkia orientalis, the putative ascogenous perfect state of C. krusei. Amounts as small as 60 to 120 ng of C. krusei target DNA were detected by dot blot hybridization with CkF1,2. It permitted the direct screening of colony blots for early identification. The CkF1,2 probe has potential value as a diagnostic reagent for identifying C. krusei.