Time-resolved fluorescence decay of a single-stranded DNA decamer d(CTGAAT5CAG), where d5 is the fluorescent base l-(β-D-2′-deoxyribosyl)-5-methyl-2-pyrimidinone, was measured and analyzed at several temperatures. The d5 base in the decamer is resolved into three states according to their fluorescence decay lifetime characteristics and temperature dependence of their associated amplitudes: fully extended and completely unstacked state, loosely associated state, and fully stacked state. These states are in slow exchange compared to their fluorescence decay rates. The population of the fully extended and completely unstacked state is small and decreases further with increasing temperature. The loosely associated state, whose fluorescence can still be efficiently quenched by other DNA bases, occupies a large portion of the conventionally defined unstacked state. Stacking enthalpy and entropy for the d5 base with thymine or cytosine bases in the DNA decamer are calculated to be −6.6 kcal/mol and −22 cal/mol·K, respectively. This work shows that fluorescent bases in DNA can be useful to the study of local conformations of bases. © 1990, American Chemical Society. All rights reserved.