Extracellular ATP is an important signaling molecule in many cell types inducing a diversity of cellular processes. In the alveoli of the lung, ATP promotes secretion of pulmonary surfactant and fluid transport. Yet, little is known how the levels of ATP in the alveoli are regulated. One mechanism involves ATP release in response to mechanical deformation of alveolar epithelial cells. In this study, we aimed at investigating ATP release at the molecular and single cell level. Therefor, we implemented an amperometric microbiosensor to measure ATP levels in close proximity (approx. 50 µm) to primary alveolar epithelial cells. In contrast to conventional bulk quantification of ATP release, such as the luciferin/luciferase-based bioluminescence assay, miniaturized biosensors enable the time-resolved measurement and determination of ATP levels with high spatial resolution. The biosensor concept for ATP detection is based on a competitive assay using glucoseoxidase and hexokinase, which are immobilized within a polymer matrix at the surface of a platinum ultramicroelectrode (diameter 50 µm). In order to position the microbiosensor, dual-electrode assemblies were used. A non-modified electrode, recording the current for oxygen reduction upon approach to the cell surface is used for positioning. A second UME, modified with the enzymatic layer, serves as biorecognition element for detecting released ATP. The calibration curve of the ATP biosensor is linear in an ATP concentration range of 0-40 µM and provides a sensitivity of 1-5 pA/µM ATP. First results of localized ATP release from stimulated alveolar epithelial type II cells will be presented.