The role of 1-pyrroline was studied via feeding experiments using rice calli cultures to gain further insight into the key steps of 2-acetyl-1-pyrroline (2AP) biosynthesis in rice. The origin of the acetyl donor was also studied through stable isotope labelled substrates. Incubation of fresh calli from a fragrant rice variety (Aychade) and a non-fragrant variety (Gladio × Fidji K2) with 1-pyrroline led to a significant increase in 2AP in both varieties. Importantly, the amount of 2AP in the non-fragrant variety could be greatly enhanced by this supplementation. When rice calli were fed with increasing levels of 1-pyrroline, 2AP levels increased accordingly. Our data show that 1-pyrroline is a limiting factor for 2AP synthesis in rice. Heat treatment of calli suggested that 1-pyrroline might be enzymatically acetylated. The presence of labelled 2AP in calli supplemented with [U-13C]glucose, sodium acetate (1,2-13C2) and sodium octanoate (1,2,3,4-13C4) suggested that these compounds are possible candidates for acetyl group-donors of 2AP, predominately in the form of intact labelled 13C2-units.