Mechanisms are required by all organisms to maintain the concentration of essential heavy metals (e.g. Zn and Cu) within physiological limits and to minimise the detrimental effects of non-essential heavy metals (e.g. Cd). Heavy-metal P-type ATPases (HMAs) are a subgroup of the P-type ATPase superfamily that may contribute to metal homeostasis in plants. We cloned and characterised a member of this family, AtHMA4, from Arabidopsis thaliana that clusters with the Zn/Co/Cd/Pb subclass of HMAs on phylogenetic analysis. Sequencing of the AtHMA4 cDNA showed that it contained the conserved motifs found in all P-type ATPases and also motifs that are characteristic of heavy-metal ATPases. Escherichia coli mutants defective in the HMAs, CopA and ZntA, were used in functional complementation studies. AtHMA4 was able to restore growth at high [Zn] in the zntA mutant but not at high [Cu] in the copA mutant, suggesting a role in zinc transport. Heterologous expression of AtHMA4 in Saccharomyces cerevisiae made the yeast more resistant to Cd but did not affect sensitivity to other metals compared with vector-transformed controls. The organ specificity of AtHMA4 was analysed in Arabidopsis and showed that AtHMA4 was expressed in a range of tissues with highest expression in roots. AtHMA4 was upregulated in roots exposed to elevated levels of Zn and Mn but downregulated by Cd. Possible physiological roles of this transporter in Arabidopsis are discussed.