Different plant organelles have high internal stores of Ca(2+) compared to the cytoplasm and could play independent roles in stress responses or signal transduction. We used a GFP fusion with the C-domain of calreticulin, which shows low-affinity, high capacity Ca(2+) binding in the ER, as a calcium-binding peptide (CBP) to specifically increase stores in the ER and nucleus. Despite the presence of a signal sequence and KDEL retention sequence, our work and previous studies (Brandizzi et al. Plant Journal 34:269-281, 2003) demonstrated both ER and nuclear localization of GFP-CBP. Under normal conditions, GFP-CBP-expressing lines had ~25% more total Ca(2+) and higher levels of chlorophyll and seed yield than wild type and GFP controls. CBP-expressing plants also had better survival under intermittent drought or high salt treatments and increased root growth. One member of the CIPK (calcineurin B-like interacting protein kinase) gene family, CIPK6, was up-regulated in CBP-expressing plants, even under non-stress conditions. A null mutation in cipk6 abolished the increased stress tolerance of CBP-transgenic plants, as well as the CBP-mediated induction of two stress-associated genes, DREB1A and RD29A, under non-stress conditions. Although this suggested that it was the induction of CIPK6, rather than localized changes in Ca(2+), that resulted in increased survival under adverse conditions, CIPK6 induction still required Ca(2+). This work demonstrates that ER (or nuclear) Ca(2+) can directly participate in signal transduction to alter gene expression. The discovery of a method for increasing Ca(2+) levels without deleterious effects on plant growth may have practical applications.