Fatty acid synthesis from Na (1-14C) acetate in leucoplasts isolated from developing seeds of Brassica compestris was found to be maximum when leucoplasts were supplied with 0.8 mM acetate, 20 mM NaHCO3, 8 mM ATP, 8 mM MgCl2, 4 mM MnCl2, 0.6 mM CoA, 1 mM NADH, 1 mM NADPH and 0.2 M sorbitol and incubated at 30°C for 2 h. The rate of fatty acid synthesis was highest at pH 8.5 In presence of 0.4 M Bistris-propane buffer and linear for upto 4 h at 30°C with 80–110 μg plastid protein. Sorbitol was an essential requirement as it prevented the rupturing of leucoplasts by osmosis. ATP and divalent cations were almost absolute requirements, whereas nucleotides, CoA and bicarbonate improved the rate of fatty acid synthesis by two to ten folds. Mg2+ and NADH were the preferred cation and nucleotide, respectively. High concentration of dithiothreltol inhibited the incorporation of (14C) acetate Into fatty acids. The system developed as above could be used for in vitro studies.