The nutritional requirement and cultural condition such as carbon and nitrogen sources, cultural temperature, initial pH, cultural time and aeration for the production of endocellular cytosine deaminase from Aspergillus fumigatus IFO 5840 were investigated. The cultural broth giving maximum cytosine deaminase yield was found to consist of 2% glucose as a carbon source and 1% yeast extract and 0.1% peptone as a nitrogen source. Optimal initial pH of the culture broth was pH 8.5 and the enzyme production in the cell usually reached a maximum after 28 hours of cultivation in the 500ml shaking flask containing 100ml broth at $30^{circ}C$. The endoenzyme production of the used strain was inhibited by inorganic nitrogen, but activated by orgainc nitrogen, yeast extract.