Substrate specificity has been pinpointed as key to understanding plant species distributions and to differentiating types of plant rarity. Substrate specific distribution patterns (e.g., calcicole and calcifuge) are particularly prevalent among bryophytes. Many authors have suggested that the effects of substrate chemistry might be most important during the establishment stage. The connection between substrate sensitivity during establishment and the degree of substrate specificity (i.e., substrate breadth) measured for adult plants in the field was explored for three rare moss species, Mielichhoferia macrocarpa, Didymodon johansenii, and Mnium arizonicum and three taxonomically allied common species, Bryum pseudotriquetrum, Didymodon rigidulus var. gracilis, and Mnium spinulosum, respectively. Substrate pH and organic matter content were determined for field samples to quantify species substrate breadths. These observed specificities were tested by comparing regeneration of gametophyte fragments on native and chemically opposite, non-native substrates. The three rare species were not consistently more substrate specific than their paired common species. Overall, establishment was higher on native substrates. The two moss species, M. macrocarpa and D. rigidulus var. gracilis, with the narrowest substrate breadths for pH and percent organic matter, were also the most sensitive to regeneration on non-native substrate. The significantly higher sensitivity of these two calcicole species in comparison with the other study species demonstrated that these calcicole species were more immediately sensitive to regeneration on non-native substrates than the calcifuge species, M. arizonicum and M. spinulosum and the calcicole species found on substrates with a wider range of organic content, B. pseudotriquetrum and D. johansenii, were less sensitive to substrate chemistry during regeneration. The use of regeneration experiments for exploring the degree and basis of species habitat specificities can help to resolve which habitat parameters physiologically limit the species. Information gained from such studies could be particularly useful for managing rare bryophyte populations.