Many drugs are weak bases and can be accumulated into liposomes in response to a pH gradient to achieve high internal drug concentrations. This study is aimed at gaining an understanding of the relationship between the retention of the fluoroquinolone antibiotic ciprofloxacin in liposomes and the intraliposomal form and location of this drug. 1H-NMR spectroscopy was used to probe the interactions experienced by ciprofloxacin following uptake into large unilamellar liposomes (LUV). It is shown that ciprofloxacin is located in the aqueous interior of the liposomes and is self-associated in the form of small stacks. It does not precipitate out of solution even though the intraliposomal ciprofloxacin concentration can exceed its solubility in aqueous solutions by almost two orders of magnitude. The results also indicate that little entrapped ciprofloxacin partitions into the inner monolayer of the LUV. As a result of the lack of precipitation and rapid exchange properties, ciprofloxacin can respond quickly to changes in electrochemical equilibria such as depletion of the pH gradient. This provides a rationale for the rapid leakage of this drug in response to serum destabilization or depletion of the pH gradient.