Nucleic acid hybridization probes are provided which comprise an N.sup.4 -(substituted amino)cytosine moiety, wherein the substituted amino group comprises a tag moiety, whereby the probe is detected. Methods of preparing probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in hybridization assays are also provided. Typical tag moieties employed with the probes of the invention are biotinyl, aminothiadiazole and fluorescein derivatives, connected to N.sup.4 -amino groups of modified cytosines of the probe through linker moieties. Probes tagged with biotin are typically detected by binding to the biotinyl moieties, through a streptavidin or avidin molecule, a reporter group which includes streptavidin or avidin and then detecting a signal due to the reporter group. Probes tagged with aminothiadiazole derivatives are typically detected by essentially the same method as those tagged with biotinyl but employing as reporter group one which binds to the derivative through a carbonic anhydrase molecule. Probes tagged with fluorescein derivatives are detected by a fluorescence spectroscopic method without binding of a reporter group to the tag.