Lifetimes of the lowest excited singlet (S₁) electronic states of various derivatives of the pyrimidine nucleobase cytosine (Cyt) were measured by the femtosecond transient absorption technique. The bases were excited in room-temperature aqueous solution at 265 nm using approximately 200 fs pump pulses from a titanium–sapphire laser system. The decay of excited-state absorption (ESA) at visible probe wavelengths was used to determine the S₁ lifetimes of a variety of modified Cyt compounds at different pH values by global fitting. Identical lifetimes were observed for Cyt and cytidine (Cyd) within experimental uncertainty, but ESA by the ribonucleoside was considerably stronger, suggesting that the ribose group increases the oscillator strength of the S₁ → S_N transition. The S₁ lifetime of the important minor base 5-methylcytosine (m⁵Cyt) is 7.2 ± 0.4 ps at pH 6.8. The same lifetime was measured for the ribonucleoside 5-methylcytidine, but sugar substitution again increased the strength of the ESA signal. Protonation of Cyd and m⁵Cyt at low pH led to a modest decrease in their S₁ lifetimes. On the other hand, deprotonation of Cyt and m⁵Cyt significantly increased the lifetime of their respective S₁ states. These trends support the intermediacy of the n,π* state localized on the carbonyl oxygen in the nonradiative decay mechanism of Cyt. Longer S₁ lifetimes were observed for 5-fluorocytosine and N-acetylcytosine. Collectively, these results illustrate the great potential of femtosecond laser spectroscopy for investigating excited-state dynamics in DNA and DNA components.