Two types of kynureninase were isolated from Neurospora crassa IFO 6068. The formation of one of them, which was separated from the inducible kynureninase by DEAE-cellulose chromatography, was independent of the presence of tryptophan in the growth medium. Ouchterlony double-diffusion analysis and immunochemical titration indicated that the constitutive-type enzyme is immunologically different from the inducible enzyme. We confirmed by a selective assay method with antiserum that the addition of tryptophan to the medium does not affect the formation of one of the enzymes (constitutive-type). The constitutive kynureninase was purified approximately 650-fold and was free of the inducible enzyme as judged by analytical gel electrophoresis. The molecular weight and optimum pH values of both enzymes are very similar. However, the constitutive enzyme shows much higher activity and affinity for L-3-hydroxykynurenine than for L-kynurenine, suggesting that the enzyme functions biosynthetically as a 3-hydroxykynureninase. Constitutive kynureninase activities were widely found in all the fungi tested, whereas the inducible enzyme activity was not present in Mucor or Rhizopus species. The inducible enzymes of all the Neurospora strains examined were shown to be immunologically identical.