The generation of 14CO2 from [1-14C]lysine by hepatic mitochondria through the saccharopine pathway is controlled by intramitochondrial concentrations of lysine, 2-oxoglutarate and NADPH. Mitochondria, isolated from rats pre-treated with glucagon, exhibited higher activities of L-lysine: 2-oxoglutarate reductase, saccharopine dehydrogenase and 2-aminoadipate aminotransferase. The flux through this pathway is stimulated in liver mitochondria after glucagon treatment. Multiple regulation of lysine oxidation in liver mitochondria confirms a complex mechanism of ‘mitochondrial activation’ by glucagon.