Regulation of cytokinin oxidase (CKX) activity in relation to enzyme glycosylation and secretion was studied in wild-type (WT) and transgenic conditionally isopentenyltransferase gene (ipt)-expressing (IPT) tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cell suspensions, calli and leaves. An increase in endogenous cytokinin content due to the tetracycline (Tc)-induced derepression of the ipt gene transcription or surface application of N6-benzylaminopurine (BA) resulted in significant enhancement of CKX activity in all these plant materials. As revealed by Concanavalin A-Sepharose 4B chromatography the cytokinin-induced enhancement of CKX activity was associated predominantly with the N-glycoform of the enzyme (10- to 15-fold increase) in calli and leaves. Application of BA to the culture media of WT and IPT cell suspensions and the derepression of the ipt by Tc substantially enhanced endogenous levels of isoprenoid cytokinins and CKX activity in both cells and the culture medium. Most CKX activity in control, BA- and Tc-treated cells was associated with the non-glycosylated form of the enzyme, whereas the majority of CKX activity in the culture media was due to the glycosylated form. The pH optimum of CKX in cells (pH 8.5) differed considerably from that in the culture medium (pH 6.0). No significant differences were found in apparent Km(iP) values of CKX between control, BA- and Tc-treated IPT cells and media or between purified glycosylated and non-glycosylated CKX. These results suggest that cytokinins induce changes in the proportions of glyco- and non-glycoforms of the enzyme in multicellular calli and leaves, and influence its secretion to the cell exterior.