The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized state was monitored by flow cytometry measuring the fluorescence of membrane-potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association with liposomes. After interaction of mitochondria with (14)C-PS containing liposomes, (14)C-PS became a substrate of PS decarboxylase, as monitored by the formation of (14)C-phosphatidylethanolamine (PE), indicating translocation of (14)C-PS to the inner membrane. The kinetics of (14)C-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared to control (state 1). In state 3, (14)C-PE formation decreased in the presence of NaN(3). Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria has not been completely elucidated. A crude mitochondrial fraction (P(2)) containing MAM, synaptosomes and myelin was prelabeled with (14)C-PS and incubated in different respiratory states. At a high respiratory rate, low-density labeled mitochondria, whose band overlaps that of synaptosomes, were obtained by centrifugation. A parallel decrease of both radioactivity and protein in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of (14)C-PS in P(2) membranes were also studied by incubating P(2) with (14)C-serine. In the resting state (14)C-PS accumulated in MAM, indicating that the transfer to mitochondria was a limiting step. In state 3 both the transfer rate of (14)C-PS and its conversion to (14)C-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism that allowed the transport of PS across the outer mitochondrial membrane.