All orchid species are associated with fungi and other organisms during their life cycles. A review of the literature on genera in the Cypripedioideae (Slipper orchids) indicated that few species of 'Cypripedium' have been studied in terms of root-associated organisms and features of root anatomy that may be related to colonization of roots by these organisms. Likewise, there are few data on the microorganisms associated with the roots of ' Paphiopedilum' and 'Phragmipedium', two subtropical/tropical orchid genera in Cypripedioideae. Field samples of roots of five ' Cypripedium' species from Manitoba and Ontario showed high levels of colonization by mycorrhizal fungi. Microorganisms isolated into sterile culture included several fungal species known to be mycorrhizal with orchids, dematiaceous fungi, including 'Phialocephala fortinii', cyanobacteria, algae and bacteria. Roots of all 'Cypripedium' species had an exodermis of suberized cell walls that may limit the ingress of most microorganisms into the cortex. Isolates of 'Epulorhiza' sp., a known mycorrhizal fungal genus, affected colonization of protocorms of 'Cypripedium reginae ' differently depending on culture substrate. A large experiment to test the effects of various root-associated organisms on the growth of sterile-raised ' C. reginae' seedlings showed that 'Epulorhiza' sp. increased shoot phosphorous and the number of roots. One bacterial isolate showed positive growth effects similar to those caused by plant growth promoting rhizobacteria (PGPRs). Many organisms, including mycorrhizal fungi, other fungi, cyanobacteria, algae and ciliated protozoans were isolated from within the multilayered epidermis (velamen) of roots collected from greenhouse-grown species of 'Paphiopedilum' and 'Phragmipedium'. Mycorrhizal fungi were also localized within the cortex. The diversity of root-associated organisms shown in this study should be taken into consideration during the commercial propagation of these genera and the reintroduction of seedlings into field sites during conservation programs.