Fluorescence excitation spectra of 2-aminopurine (2AP) incorporated into single-stranded DNA di-and trinucleotides, as well as into single- and double-stranded pentanucleotides, have been measured as a function of temperature from 5 to 65°C. Spectral shifts have been precisely quantitated through difference spectroscopy and spectral fits. G(2AP)C and C(2AP)G oligonucleotides have relatively blue-shifted excitation spectra (especially the former) compared to the 2AP free base. The position of the excitation peak of 2AP free base is temperature independent, those of (2AP)T, G(2AP)C, C(2AP)G and TT(2AP)TT shift about 0.4 nm to the blue from 5 to 65 0C, though the spectra of the G-C-containing oligomers also change shape. The temperature dependence of the A(2AP)T spectral position is 2.5-times stronger, and just rises to that of the free base at high temperature. On the other hand, the decrease of yield with increasing temperature is smallest for A(2AP)T, even compared to the free base. The dominant effect when A neighbors 2AP appears to be temperature-dependent stacking with accompanying energy transfer, while in G- and C-containing trinucleotides a temperature-independent interaction keeps the 2AP excitation spectrum blue-shifted. The effect of double strand formation appears to be small compared to stacking interactions. These spectra can be useful in identifying base neighbors and structures of 2AP in unknown 2AP-labeled DNA.