BackgroundAsparagine is one of the most dominant organic nitrogen compounds in phloem and xylem sap in a wide range of plant species. Asparaginase (ASNase; EC, 3.5.1.1) catabolizes asparagine into aspartate and ammonium; therefore, it is suggested to play a key role in asparagine metabolism within legume sink organs. However, the metabolic fate of asparagine in source and sink organs during rice seed production remains to be elucidated. Therefore, the main objective of this study is to investigate the asparagine metabolism in a temporal and spatial manner during rice seed production.ResultsFor this purpose, the expression of genes involved in asparagine catabolism, such as asparaginase1 (OsASNase1) and 2 (OsASNase2), were quantitatively measured, and contents of asparagine, aspartate and ammonium ions were determined in sink and source organs during spikelet ripening. Quantitative real-time PCR and in situ localization studies determined that OsASNase2 is expressed in the dorsal vascular bundles and nucellar projection of developing grains, as well as in mesophyll and phloem companion cells of senescent flag leaves. Amino acid measurements revealed that the aspartate concentration is higher than asparagine in both source and sink organs.ConclusionThis work suggests that asparaginase dependent asparagine catabolism occurred not only in sink but also in source organs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-017-0143-8) contains supplementary material, which is available to authorized users.