A key step of the intrinsic apoptotic pathway is the assembly of the apoptosome complex. The apoptosome components are well known, however, the physiology of its assembly at cellular level is still poorly defined. The aim of this work was to study the sub-cellular distribution of the apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) before and after triggering apoptosis in single somatotrophs. Somatotrophs are the subject of extensive pituitary tissue remodeling in different physiological situations, where the quality and the number of pituitary cells are determined by cell proliferation and by apoptosis. We show here that 2 h after triggering apoptosis with rotenone Apaf-1 redistributed to the proximity of mitochondria. In addition, the degree of co-localization between Apaf-1 and fluorescently labeled caspase-9 significantly increased in the same time period. Furthermore, we show here for the first time in single cells that the co-localization between Apaf-1 and cytochrome-c increased only transiently, indicating a transient interaction between cytochrome-c and Apaf-1 during the activation of apoptosis in these cells.