In this study, we synthesized small-molecule, water-soluble, fluorescein-containing ureido compounds 6 and 8 as target receptors for cytosine free bases and then investigated the binding of cytosine free bases with the receptors using 15N NMR spectroscopy and partially labeled cytosine-2,4-13C-1,3,4-15N-cytosine. Binding with the receptor 6a (the disodium form of 6) caused the chemical shift of the nitrogen atom of the amino group of cytosine to move downfield; binding of the receptor 8a (the disodium form of 8), which is possessing no corresponding aryl nitrogen atom, had no effect on this signal. Fluorescence spectroscopy revealed that binding of cytosine and its derivatives led to quenching of the fluorescence of receptor 6a; in contrast, the quenching of receptor 8a was only slightly affected by cytosine. Because the fluorescence of 6a was not quenched by either deoxycytidine or uracil, it appears that this receptor is a specific for cytosine among the DNA bases. We used the fluorescence of 6a to measure the apparent binding constants for various cytosine derivatives, including the anticancer prodrug 5-fluorocytosine. Receptor 6a is the first small-molecule, water-soluble fluorescent receptor for the specific binding of cytosine free bases in aqueous solution.