Regulation of Ca(2+) transporters is a vital component of signaling. The Arabidopsis H(+)/Ca(2+) exchanger CAX1 contains an N-terminal autoinhibitory domain that prevents Ca(2+) transport when CAX1 is heterologously expressed in yeast. Using a yeast screen, we have identified three different proteins that activate CAX1. One of these, CXIP1 (CAX-interacting protein-1; 19.3 kDa) has amino acid similarity to the C terminus of PICOT (protein kinase C-interacting cousin of thioredoxin) proteins. Although PICOT proteins are found in a variety of organisms, a function has not been previously ascribed to a plant PICOT protein. We demonstrate that CXIP1 activated the CAX1 homolog CAX4, but not CAX2 or CAX3. An Arabidopsis homolog of CXIP1 (CXIP2) weakly activated CAX4, but not CAX1. In a yeast two-hybrid assay, CXIP1 interacted with the N terminus of CAX1. In competition analysis, CXIP1 and a CAX1 N-terminal peptide appeared to bind to similar N-terminal domains of CAX1. Chimeric CAX3 constructs containing the N terminus of CAX1 were activated by CXIP1. In Arabidopsis, CXIP1 transcripts, like CAX1, accumulated in response to different metal conditions. This work thus characterizes a new class of signaling molecules in plants that may regulate CAX transporters in vivo.