Objective: Autocrine ATP signaling acting at P2Y₂ receptors has been implicated in gradient sensing by mouse neutrophils (Chen et al. 2006). Here, we tested whether autocrine purinergic signaling is important for the navigation of resident mouse peritoneal macrophages along a chemotactic gradient. Extracellularly released ATP is degraded to ADP and adenosine, giving rise to at three feedback loops terminating at P2Y₂ (ATP), P2Y₁₂ (ADP) and adenosine receptors. Methods: We established a macrophage chemotaxis assay using the Ibidi m-slide chemotaxis slide and the chemoattractant complement component C5a. In addition, we investigated chemotaxis towards C5a in vivo. Results: Wild-type (WT) control macrophages migrated robustly towards C5a, whereas chemotaxis in vitro and in vivo was inhibited by apyrase. Thus, autocrine ATP signaling is essential for efficient chemotactic navigation. The inhibitory action of apyrase could not be reproduced using P2Y₂ knockout (P2Y₂-/-) or P2Y₁₂12-/- macrophages or a non-specific adenosine receptor inhibitor. However, chemotaxis could be blocked by inhibiting all three feedback loops. Furthermore, we found that stimulation of each of these feedback loops induced lamellipodial membrane protrusions, major determinants of motility and direction. Conclusion: Our data suggest a model whereby autocrine purinergic signaling is required to amplify and translate chemotactic cues into directed motility. References: Chen, Y., Corriden, R., Inoue, Y., Yip, L., Hashiguchi, N., Zinkernagel, A., Nizet, V., Insel, P.A. & Junger, W.G. 2006. Science 314, 1792-1795.