Understanding the interactions of small molecules like antibiotics with RNA is a prerequisite for the development of novel drugs. In this study we address structural and thermodynamic features of such interactions by using a simple model system: the binding of the highly charged antibiotic neomycin B to a short hairpin RNA molecule. Nucleotide A16, which acts as a flap over the neomycin B binding pocket, was substituted by the fluorescent adenine analogue 2-aminopurine (2-AP). Steady-state and time-resolved fluorescence measurements were complemented by UV-melting and circular dichroism studies. The binding of neomycin B at three sites was found to have a strong inverse correlation with Na(+) concentration. For the highest-affinity site, both fluorescence and UV absorption experiments were consistent with a model assuming at least three neomycin NH(3) (+) groups participating in addition to hydrogen bonds in electrostatic interactions with the RNA. The variation of fluorescence intensity and lifetime upon neomycin B binding indicated unstacking of 2-AP16 from neighbouring bases as it flipped over the binding pocket. RNA conformational changes upon binding of the antibiotic were confirmed by circular dichroism. The two weaker binding sites were characterized as unspecific binding to the aptamer, while the high-affinity binding event was shown to be highly specific even at high ionic concentration. In addition, 2-AP was confirmed to be a noninvasive fluorescent probe; it serves as a sensitive spectroscopic tool to investigate details of the interactions between small molecules and RNA.