Zinc is an essential micronutrient in humans and plants, where it serves structural roles and functions as a catalytic element in hundreds of proteins. Zinc deficiency is estimated to affect more than two billion people globally. Developing plants that accumulate more zinc in their edible parts has been proposed as a plausible solution to combat zinc deficiency. Plants take up zinc from the soil into the root from where it is transported throughout the plant to the developing tissues and seeds. Through the journey from root to seed, it needs to cross several physical barriers. Such barriers are found in the apoplast, in the form of deposited cell wall polymers, and in the symplast, in the form of biological membranes. Transport of cations across the plasma membrane into the symplast is against the electrical gradient and, therefore, active transport is required. In Arabidopsis thaliana (A. thaliana), two heavy metal ATPases (AtHMA2 and AtHMA4 from the P1B2 subgroup of P‐type ATPases) are involved in the active export of zinc from the symplast of root pericycle cells to the apoplastic xylem, and in the seed from the symplast of the maternal seedcoat to the apoplast surrounding the filial tissues. The C‐termini of P1B2 ATPases are elongated and have been suggested to serve as zinc sensors and control transport activity negatively. The purpose of this work was to examine the physiological effects of partially removing C‐terminal residues of two P1B2 ATPases: AtHMA4 from A. thaliana and HvHMA2 from Hordeum vulgare. For this purpose, mutant plants encoding C‐terminally truncated protein were isolated and phenotyped. In H. vulgare, HvHMA2 was found to be expressed in cells facing the apoplastic space separating the maternal and filial tissues. A mutant of H. vulgare that expresses a version of HvHMA2 devoid of 99 C‐ terminal residues (Hvhma2Δ99) had increased zinc in the seed, whereas deleting 33 C‐terminal residues (to produce Hvhma2Δ33) was not sufficient for such an effect. These results were confirmed in a heterologous system where Hvhma2Δ99, but not Hvhma2Δ33, was able to complement a zinc‐ sensitive yeast strain. This would indicate that partial removal of the negative regulatory domain of HvHMA2 results in increased zinc export to the seed. In A. thaliana, two mutants, Athma4Δ314 and Athma4Δ393, devoid of 314 and 393 residues, respectively, were investigated. When crossed into the Athma2 knockout background, only Athma4Δ314 supported growth under normal conditions but displayed a smaller plant phenotype. Investigating the seed zinc distribution of Athma4Δ314 revealed it to be identical to that of an Athma4 knockout. A distinct dual subcellular localization to the plasma membrane and internal membranes was observed for Athma4Δ314, whereas Athma4Δ393 completely mislocalized to internal membranes. This would indicate that the stretch of amino acids between the two mutants is involved in the correct subcellular targeting of the transporter to the plasma membrane.