In eucaryotes, glycolytic enzymes are classically regarded as being localised in the cytosol. Recently, we have shown that part of the cellular pool of the glycolytic enzyme, enolase, is tightly associated with the mitochondrial surface in the yeast Saccharomyces cerevisiae (N. Entelis, I. Brandina, P. Kamenski, I.A. Krasheninnikov, R.P. Martin and I. Tarassov, A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae, Genes Dev. 20 (2006) 1609-1620). Here, using enzymatic assays, we show that all glycolytic enzymes are associated with mitochondria in yeast, to extents similar to those previously reported for Arabidopsis cells. Using separation of mitochondrial complexes by blue-native/SDS-PAGE and coimmunoprecipitation of mitochondrial proteins with anti-enolase antibodies, we found that enolase takes part in a large macromolecular complex associated to mitochondria. The identified components included additional glycolytic enzymes, mitochondrial membrane carriers, and enzymes of the TCA cycle. We suggest a possible role of the enolase complex in the channeling of pyruvate, the terminal product of glycolysis, towards the TCA cycle within mitochondria. Moreover, we show that the mitochondrial enolase-containing complex also contains the cytosolic tRNA(CUU)Lys, which is mitochondrially-imported, and its presumed import carrier, the precursor of the mitochondrial lysyl-tRNA synthetase. This suggests an unsuspected novel function for this complex in tRNA mitochondrial import.