The qualitative composition and dynamics of cytokinins in the fronds and rhizomes of perennial ferns Dryopteris filix-mas and Polystichum aculeatum have been investigated using high-performance liquid chromatography in combination with mass spectrometry. Sporophytes were studied at the stages of intensive vegetative growth (April), formation of sporangia (May) and sporulation (June). Plants of P. aculeatum were also analyzed at the stage of winter vegetation (February). The accumulation of trans-zeatin in fronds of P. aculeatum was revealed at the intensive growth stage, whereas in D. filixmas, the increase in this cytokinin content was observed during the formation of sporangia. The level of zeatin riboside increased in fronds and rhizomes of both fern species at the stage of sporulation. The cessation of the ferns intensive growth involved the accumulation of conjugate-zeatin-O-glucoside. At certain stages of sporophyte development, isopentenyl-type cytokinins were detected: in D. filix-mas, low levels of isopentenyladenine-during sporulation, and in P. aculeatum, quite significant amounts of isopentenyladenosine and isopentenyladenine-in fronds at the stage of intensive growth and in rhizomes-during sporangia formation. In P. aculeatum fronds, active free cytokinins-zeatin and zeatin riboside-were accumulated during the winter vegetation indicating that they were involved in the maintenance of the plant photosynthetic activity under unfavorable conditions. The root system of both fern species was characterized by a lower level of cytokinins as compared to the aerial part. The dynamics of the spectrum and content of cytokinins in the fern organs was species-specific and indirectly testified to the involvement of these phytohormones in growth and development control. The features of differences and similarities of the regulatory role of cytokinins in ferns and seed plants are discussed.