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The effect of sulfur (S) fertilisation on oil accumulation, acetyl-CoA concentration, and activity of acetyl-CoA carboxylase (EC 6.4.1.2) was determined in the developing seeds of rapeseed (Brassica campestris L. cv. Pusa Gold) grown in the field with and without S. The period between 14 and 35 days after flowering (DAF) was identified as the active period of oil accumulation in the developing seeds of rapeseed. The accumulation of oil was preceded by a marked rise in acetyl-CoA carboxylase activity and acetyl-CoA concentration, which declined rapidly when oil accumulation reached a plateau. Starch and soluble sugar content decreased, while protein content increased during the period of active oil accumulation in the developing seeds (i.e. 14–35 DAF). Sulfur fertilisation significantly (P < 0.05) enhanced the oil accumulation in the developing seeds at all the growth stages except at 7 DAF. The increase in the oil content was 13.0–52.0% with S fertilisation over the control treatment. Sulfur fertilisation also increased acetyl-CoA concentration, acetyl-CoA carboxylase activity, and soluble protein, sugar, and starch content in the developing seeds. It is suggested that the increase in the oil content with S fertilisation may be associated with increases in acetyl-CoA carboxylase activity through the enhancement of acetyl-CoA concentration. Further, the increased sugar content due to S fertilisation provided enough carbon sources for oil biosynthesis.