There is strong evidence for a purinergic signalling system in the inner ear which regulates auditory sensitivity. This study describes the terminating mechanism for purinergic signalling in the cochlear endolymphatic compartment via ecto-nucleotidases. Exogenous ATP was introduced into the scala media (SM) of the isolated, perfused guinea-pig cochlea, and the effluent was assayed for the adenine nucleotide metabolites by reverse-phase HPLC. Tissue viability was confirmed by fluorescence imaging of cochlear tissues. Extracellular ATP degradation to adenosine was Ca2+/Mg2+ dependent, and was not affected by inhibitors of intracellular ATPases and non-specific alkaline phosphatase. High azide concentration (5 mM) and suramin produced an inhibitory effect on ATP hydrolysis, consistent with inhibition of E-type ATPase activity. The Vmax of ATP hydrolysis (2564 mumol min-1 SM-1) was indicative of high ecto-ATPase activity. Our results support the role of ecto-nucleotidases as a principal mechanism for termination of purinergic signalling within SM, a compartment of the cochlea showing considerable P2X receptor expression.