As part of the conservation programme for Cypripedium calceolus in England, seedlings have been produced in axenic culture and grown on in pots prior to planting out at putatively suitable locations in northern England; the success of these introductions is expected to be dependent on the presence of appropriate mycorrhizal fungi at the introduction sites and the re-establishment of mycorrhizal associations by the introduced plants. To investigate mycorrhizal function, leaf samples of C. calceolus and associated non-orchid species were collected from the native wild site and from plants at three of the introduction sites, and screened for enrichment in isotopes of carbon (C), nitrogen (N) and hydrogen (H) that are indicative of an exchange of matter between fungi and orchids in orchid mycorrhizal associations. Total N concentration was also measured. At all sites, plants of C. calceolus were enriched in ²H and, to a lesser extent, ¹⁵N relative to the associated species and had higher concentrations of total N. There was no apparent enrichment in ¹³C. Together, these results indicate that all the screened individuals of C. calceolus (wild and introduced) are likely to have formed a mycorrhizal association with a rhizoctonia. Thus, the axenically produced seedlings may have established a mycorrhizal relationship after introduction with the same type of fungus utilized by C. calceolus elsewhere in its range.