The 5-hydroxytryptamine 3 (5-HT3) receptor belongs to the pentameric ligand-gated cation channel superfamily. Humans have five different 5-HT3receptor subunits: A to E. The 5-HT3receptors are located on the cell membrane, but a previous study suggested that mitochondria could also contain A subunits. In this article, we explored the distribution of 5-HT3receptor subunits in intracellular and cell-free mitochondria. Organelle prediction software supported the localization of the A and E subunits on the inner membrane of the mitochondria. We transiently transfected HEK293T cells that do not natively express the 5-HT3receptor with an epitope and fluorescent protein-tagged 5HT3A and 5HT3E subunits. Fluorescence microscopy and cell fractionation indicated that both subunits, A and E, localized to the mitochondria, while transmission electron microscopy revealed the location of the subunits on the mitochondrial inner membrane, where they could form heteromeric complexes. Cell-free mitochondria isolated from cell culture media colocalized with the fluorescent signal for A subunits. The presence of A and E subunits influenced changes in the membrane potential and mitochondrial oxygen consumption rates upon exposure to serotonin; this was inhibited by pre-treatment with ondansetron. Therefore, it is likely that the 5-HT3receptors present on mitochondria directly impact mitochondrial function and that this may have therapeutic implications.