Transcription of the cucumber hpr-A gene is responsive to cytokinin and light. To investigate the molecular basis for transcriptional regulation by cytokinin, we have identified DNA sequences and proteins that may be involved in the regulation of hpr-A gene expression. Transient expression assays in etiolated cucumber cotyledons indicate that the 315 bp fragment (−382 to −67) contains sequences necessary for cytokinin responsiveness of the luciferase reporter gene. Band shift assays detected cytokinin-enhanced and -reduced protein binding sites in a 97 bp fragment (−382 to −285) upstream of the hpr-A gene. DNase I footprinting identified two protein-protected sites, a 15 bp sequence, 5′-AAATGACGAAAATGC-3′, that contains an as-1 TGACG motif found in other plant promoters, and a 13 bp sequence, 5′-AAGATTGATTGAG-3′, of unknown function. Two-dimensional band shift analysis of the cytokinin-responsive DNA protein complex revealed the presence of six DNA protein interactions. Band shift assays showed that cytokinin and light have different effects on the interaction of nuclear proteins to the 97 bp fragment of the hpr-A gene. These data suggest that cytokinin and light do not share identical signal transduction pathways in regulating hpr-A gene expression.