We have determined the effect of lactate on the translocation of GLUT 1 and GLUT4 and on the myocardial uptake and phosphorylation of the glucose analogues 2-deoxy-D-glucose (DG) and 2-F-18-fluoro-2-deoxy-D-glucose ((18)FDG). The involvement of phosphatidyl-inositol-3-kinase (PI3K) in this translocation was determined using wortmannin. Hearts from fed and fasted male Wistar rats were perfused in the presence of 11 mM glucose 10 mM lactate for two hours and the distribution of glucose transporters was determined using Western blot techniques. Two other groups of hearts from fed animals were perfused in the presence of 11 mM glucose 10 mM lactate for two hours followed by perfusion for a further 30 minutes in the presence of 4 mM 2-deoxy-D-glucose. Using P-31 NMR spectroscopy, the accumulation of 2-deoxy-D-glucose-6-phosphate (DG6P) was monitored over time. Another group of hearts from fed animals was initially perfused in the presence of 11 mM glucose for 100 minutes and then the perfusate was changed to 11 mM glucose + 10 mM lactate for a further 120 minutes. Using PET, the accumulation of 2-F-18-fluoro-deoxy-D-glucose-6-phosphate ((18)FDG6P) was monitored throughout the whole protocol. Lactate induced the translocation of both GLUT1 and GLUT4 to the plasma membrane (from 67 +/- 1% to 82 +/- 2% and from 16 +/- 1% to 28 +/- 2%, respectively (P < 0.05)) in hearts from fed animals; similar translocations were observed in hearts from fasted animals. Wortmannin did not inhibit the translocation of either GLUT1 or GLUT4. Glucose transporter translocation was accompanied by a significant inhibition of DG6P accumulation (4.24 +/- 0.68 vs. 1.50 +/- 0.38; P < 0.001) and a decrease in the rate of (18)FDG6P accumulation. In conclusion, lactate causes translocation of GLUT1 and GLUT4 to the plasma membrane, via a non-PI3K-mediated pathway. Despite this externalisation of the GLUT transporters, a marked decrease in the accumulation of both DG6P and (18)FDG6P was observed.