In this study investigated the effect of cytosine deaminase activity from Aspergillus fumigatus IFO 5840 by cytosine analogues. The results were as follows. The enzyme was strongly inibited by 2-thiouracil, 2-thiocytosine, 6-azacytosine and 2-mercaptopyrimidine. The half inhibitory concentration(HIC) of 2-thiocytosine and 6-azacytosine on cytosine deaminase was 0.80mM and 1.15mM, respectively. The enzyme was inhibited at a certain level by addition of 2-thiocytosine immediately, but was maintained to some extend under the inhibited state by 6-azacytosine in proportion to reaction time. Regardless of kinds of substrate such as cytosine and 5-fluorocytosine, 2-thiocytosine and 6-azacytosine showed action as inhibitors, 2-thiocytosine inhibited cytosine deaminase activity about twice as strong as 6-azacytosine. The enzyme, when cytosine was used as a substrate, was revealed the pattern of competitive inhibition by 2-thiocytosine and 6-azacytosine, The ki value for these compounds was 4.5$ imes$10-4M and 1.756$ imes$10-3M, respectively. At this point, the Hill coefficient for cytosine, 2-thiocytosine and 6-azacytosine was 1.80, 1.81 and 2.45, respectively.