Based on the assumption that C substance has a cytosine ring bonded with a sugar, various derivatives were prepared to determine the position of sugar bonding. Deami-nation of C substance with nitrous acid and hydrolysis of its product afforded uracil. Methylation of C substance with dimethyl sulfate and hydrolysis of its product gave N, 3-dimethylcytosine, while methylation with diazomethane, followed by hydrolysis afforded 3-methylcytosine. These experimental facts indicated that neither the nitrogen in 3-position of cytosine nor the primary amino group in 4-position of the cytosine ring in the C substance had any substituent. Since the ultraviolet spectrum of the C substance was similar to that of cytidine, it was assumed that the C substance had a sugar bonded to the 1-position of its cytosine ring.