Transforming Growth Factor- (TGF-) has been implicated as a key factor in mediating many cellular processes germane to disease pathogenesis, including diabetic vascular complications. TGF- alters cytosolic [Ca²⁺] ([Ca²⁺]_c) signals, which in some cases may result from the down-regulation of the IP₃ receptor Ca²⁺ channels (IP3R). Ca²⁺ released by IP3Rs is effectively transferred from ER to the mitochondria to stimulate ATP production and to allow feedback control of the Ca²⁺ mobilization. To assess the effect of TGF- on the ER-mitochondrial Ca²⁺ transfer we first studied the [Ca²⁺]_c and mitochondrial matrix Ca²⁺ ([Ca²⁺]_m) signals in single pre-glomerular afferent arteriolar smooth muscle cells (PGASMC). TGF- pretreatment (24h) decreased both the [Ca²⁺]_c and [Ca²⁺]_m responses evoked by AII or endothelin. Strikingly, the [Ca²⁺]_m signal was more depressed than the [Ca²⁺]_c signal and was delayed. In permeabilized cells, TGF- pretreatment attenuated the rate but not the magnitude of the IP₃-induced [Ca²⁺]_c rise, yet caused massive depression of the [Ca²⁺]_m responses. ER Ca²⁺ storage and mitochondrial uptake of added Ca²⁺ was not affected by TGF-. Also, TGF- had no effect on mitochondrial distribution and on the ER-mitochondrial contacts assessed by two-photon NAD(P)H imaging and electron microscopy. Down-regulation of both IP3R1 and IP3R3 was found in TGF--treated PGASMC. Thus, TGF- causes uncoupling of mitochondria from the ER Ca²⁺ release. The sole source of this would be suppression of the IP3R-mediated Ca²⁺ efflux, indicating that the ER- mitochondrial Ca²⁺ transfer depends on the maximal rate of Ca²⁺ release. The impaired ER-mitochondrial coupling may contribute to the vascular pathophysiology associated with TGF- production.