Abstract  While mitochondrial Ca2+ homeostasis has been studied for several decades and many of the functional roles of Ca2+ accumulation within the matrix, at least in part, clarified, the possibility of modulation of the organelle functions by cAMP remains largely unknown. In this contribution we briefly summarize the key aspects of Ca2+ and cAMP signalling pathways in mitochondria. In particular, we focus on recent findings concerning the discovery of an autonomous cAMP toolkit within the mitochondrial matrix and its role in controlling mitochondrial ATP synthesis. A description of the main methods presently available to measure Ca2+ and cAMP in mitochondria of living cells with genetically encoded probes is also presented. This article is protected by copyright. All rights reserved