Peatland-inhabiting testate amoebae are sensitive indicators of substrate-moisture conditions and have increasingly been used in palaeohydrological studies. However, to improve accuracy of testate-amoeba-based hydrological inferences, baseline ecological data on rare taxa, a larger geographic network of calibration sites, and incorporation of long-term estimates of water-table depth are needed. Species–environment relationships at 369 sites from 31 peatlands in eastern North America were investigated. Long-term estimates of water-table depth were obtained using the method of polyvinyl (PVC) tape-discolouration. Transfer functions were developed using a variety of models, and validated through jackknifing techniques and with an independent dataset where water-table depths were directly measured throughout the growing season. Results indicate that mean annual water-table depth can be inferred from testate amoeba assemblages with a mean error of 6 to 8 cm, although there is a slight systematic bias. All transfer function models performed similarly and produced similar reconstructions on a fossil sequence. In a preliminary effort towards development of a comprehensive North American calibration dataset, data from this study were combined with previous studies in Michigan and the Rocky Mountains (n = 650). This combined dataset had slightly larger mean errors of prediction (8–9 cm) but includes data for several rare taxa. Copyright © 2007 John Wiley & Sons, Ltd.