The products of the tuberous sclerosis complex (TSC) genes, hamartin and tuberin (TSC1 and 2), form a heteromer which represses the kinase mammalian target of rapamycin (mTOR). Loss of TSC1 or 2 results in diseases characterised by loss of cell cycle control, including TSC and lymphangioleiomyomatosis. As tuberin has multiple signalling inputs including phosphatidylinositide-3-OH kinase, mitogen activated protein kinase and adenosine monophosphate kinase we postulated tuberin would have multiple protein interactions governed by subcellular localisation and cellular status and examined this in primary human airway smooth muscle cells. Using immunofluorescence and confocal microscopy tuberin was detected in cytoplasm, nucleus, nucleoli, and mitochondria. Fractionation of synchronised airway smooth cells showed that tuberin enters the nucleus in late G₁ and passage through the cell cycle is necessary for nuclear entry. Deletion constructs showed localisation sequences for the nucleus between amino acids 1351-1807, for mitochondria between 901-1350 and for cytoplasmic speckles between 1-450. Using fluorophore-tagged proteins we observed fluorescence resonance energy transfer between tuberin and hamartin within these speckles, indicating a direct interaction between the proteins at this site. The observations that tuberin is localised to mitochondria and translocated to the nucleus in G₁ are novel and consistent with interactions with proteins within multiple signalling pathways. Dynamic re-localisation of tuberin may control these interactions to integrate these pathways. As tuberin has potential roles in proliferation, migration and cell phenotype it therefore warrants further investigation in diseases categorised by abnormalities in airway smooth muscle.