The phytohormones cytokinins and auxin have been known as theprincipal regulators of plant development for a long time. In in vitroregeneration assays, the presence of high auxin-to-cytokininconcentration ratio in media induces root regeneration from variousplant tissues. On the other hand, if the auxin-to-cytokinin ratio is low,shoots are formed. The process of in vitro shoot regeneration is accompanied by strongactivation of cytokinin signalling via AHK4 receptor and induction ofshoot-specific homeodomain regulator WUSCHEL specifically in theregenerating organs. Exogenous but also endogenous cytokininsinfluence both the initiation of newly formed organs as well as thepace of organ developmental sequence. The role of cytokinins,importance of cytokinin biosynthesis and metabolism in the control ofin vitro shoot regeneration will be discussed.