The tautomeric preferences of cytosine and its derivatives substituted at position 5 (R = CH3, propynyl, Cl, and Br) have been analyzed both in the gas phase and in aqueous solution by using a combination of state-of-the-art theoretical methods. It is found that 5- substitutions do not alter dramatically the tautomeric preferences of cytosine in gas phase or aqueous solution. The Hoogsteen-type hydrogen-bonding and stacking properties of the imino form of cytosine and its substituted derivatives are examined in light of the results determined by using ab initio quantum mechanical and density functional calculations. It is found that imino cytosines, and especially its 5-propynyl and 5-Br derivatives show very good stacking in triplexes. The impact of the results in the design of new pyrimidines with ambiguous Hoogsteen pairing ability for the stabilization of triple helices is discussed.