Solanum tuberosum Cytokinin Riboside Phosphorylase (StCKP1), catalyses the interconversion of the N9-riboside form of the plant hormone cytokinin (CK), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as CK-binding activity in extracts of tuberising potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide, and motifs for purine nucleoside phosphorylase activity. Expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted to ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted to their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.