Fatty acid synthesis from Na [1-14C] acetate in leucoplasts isolated from developing seeds of Brassica campestris was completely dependent on exogenous supply of ATP. None of the intermediates of glycolysis or pentose phosphate pathway tested could replace ATP in the reaction mixture. In absence of exogenously supplied ATP, maximum activity was obtained with glu-6-P (68%) followed by fru-6-P (50%) and PEP (44%), respectively. With other intermediates as energy sources, the activity ranged from 1 to 38%. In complementary experiments (presence of ATP), none of the metabolites gave activity higher than the ATP control activity. Under optimum conditions for fatty acid synthesis from acetate, Brassica leucoplasts readily utilized labelled glucose as the substrate for fatty acid synthesis. Omission of NADH and NADPH individually from the reaction mixtures containing labelled glucose resulted only in 46 and 20% loss in activity, respectively, compared to the corresponding losses of 56 and 50%, when labelled acetate was used as the substrate. Similarly, deletion of ATP from the reaction mixture containing glucose as the substrate decreased the rate of fatty acid synthesis by about 65%, while the corresponding decrease with acetate as the substrate was 96%. Inclusion of 5 mM cold acetate, pyruvate, malate and glu-6-P in the reaction mixture containing glucose as the labelled substrate reduced label incorporation into fatty acids by 38 to 69%, maximum reduction being observed with pyruvate followed by glu-6-P, acetate and malate, respectively. With labelled acetate as the substrate, maximum reduction in label incorporation was obtained with cold glucose (5 mM) followed by glu-6-P, pyruvate and malate, respectively. The study demonstrated the operation of complete glycolytic pathway in Brassica leucoplasts, allowing the plastids to use glucose as a source of carbon, reducing power and energy for fatty acid synthesis.