Addition of oleoyl-CoA (1 microM), or other acyl-CoA thioesters with a chain length of C(16) or greater, to oilseed rape plastids (Brassica napus L.) inhibited the rate of D-glucose 6-phosphate (Glc6P) uptake by 70% after 2 min. The IC(50) value for oleoyl-CoA inhibition of the transporter was approx. 0.2-0.3 microM. Inhibition was alleviated by the addition of acyl-CoA binding protein (ACBP) or BSA at slightly higher concentrations. Oleic acid (5-25 microM), Tween 40 (10 microM), Triton-X 100 (10 microM) and palmitoylcarnitine (5 microM) had no effect on Glc6P uptake. The uptake of [1-(14)C]Glc6P occurred in exchange for P(i), 3-phosphoglycerate or Glc6P at a typical rate of 30 nmol Glc6P/min per unit of glyceraldehyde-3-phosphate dehydrogenase (NADP(+)). The K(m(app)) of the Glc6P transporter for Glc6P was 100 microM. Neither CoA (0.3 mM) nor ATP (3 mM) inhibited Glc6P uptake, but the transporter was inhibited by 72% when ATP and CoA were added together. This inhibition was attributable to the synthesis of acyl-CoA thioesters, predominantly oleoyl-CoA and palmitoyl-CoA, by long-chain fatty acid-CoA ligase (EC 6.2.1.3) from endogenous fatty acids in the plastid preparations. Acyl-CoA thioesters did not inhibit the uptake of [2-(14)C]pyruvate or D-[1-(14)C]glucose into plastids. In vivo quantities of oleoyl-CoA and other long-chain acyl-CoA thioesters were lower than those for ACBP in early cotyledonary embryos, 0.7+/-0.2 pmol/embryo and 2.2+/-0.2 pmol/embryo respectively, but in late cotyledonary embryos quantities of long-chain acyl-CoA thioesters were greater than ACBP, 3+/-0.4 pmol/embryo and 1.9+/-0.2 pmol/embryo respectively.