Calcium-labelled (44Ca) uptake and transport under in vitro GreenTray® temporary immersion bioreactor conditions have been studied related to aeration conditions. For this aim, Rootpac®-20 (RP-20) and Garnem® (G × N) were selected as two main rootstocks used in peach production. Two transpiration conditions, aerated and unaerated, were established for each plant material. 44Ca location, plant development and foliar stomata surface were measured after the in vitro culture period. The results showed that aeration improved Ca transport within the shoot, but it did not enhance Ca uptake by the roots. Regarding plant material, G × N presented a better Ca uptake capacity and concentration. The findings suggest that Ca uptake in the roots is a precise process that is influenced by transpiration. However, it was observed that transpiration and thus the water flux is not the only force promoting Ca uptake by roots. Furthermore, the transport of Ca to the shoot was primarily determined by transpiration, indicating that water flux plays a crucial role in the aboveground movement of Ca. The study also revealed distinct behaviors in Ca uptake and allocation between the different peach rootstocks, emphasizing the importance of considering these factors in the selection process of rootstocks. These findings contribute to our understanding of the mechanisms involved in Ca uptake and transport in peach rootstocks under in vitro conditions. They provide valuable insights for rootstock selection processes and highlight the need for further research in this area.Research sponsored by 44CaPeach (PID2019-111583RR-I00) Project (“Ministerio de Ciencia e Innovación”) and IRTA. Authors would like to also thank European Social Found (ESF) “ESF invest in your future” for Ph.D. grant PRE2020-095467 (Carrasco-Cuello, F) and Sandra Franquesa and Maria Casanovas for the technical support.info:eu-repo/semantics/publishedVersio