Cytokinin hormones are important regulators of development and environmental responses of plants that execute their action via the molecular machinery of signal perception and transduction. The limiting step of the whole process is the availability of the hormone in suitable concentrations in the right place and at the right time to interact with the specific receptor. Hence, the hormone concentrations in individual tissues, cells, and organelles must be properly maintained by biosynthetic and metabolic enzymes. Although there are merely two active cytokinins, isopentenyladenine and its hydroxylated derivative zeatin, a variety of conjugates they may form and the number of enzymes/isozymes with varying substrate specificity involved in their biosynthesis and conversion gives the plant a variety of tools for fine tuning of the hormone level. Recent genome-wide studies revealed the existence of the respective coding genes and gene families in plants and in some bacteria. This review summarizes present knowledge on the enzymes that synthesize cytokinins, form cytokinin conjugates, and carry out irreversible elimination of the hormones, including their phylogenetic analysis and possible variations in different organisms.