RNA polymerase I (pol I) transcribes the repeated genes that encode the precursor of 17-18, 5.8, and 25-28 S ribosomal RNA (rRNA). Pol I transcription is up-regulated in growing cells and down-regulated in quiescent cells, presumably reflecting the demand for ribosomes and protein synthesis. However, the signal transduction pathways responsible for pol I regulation are poorly understood. We tested the effects of exogenously applied plant hormones on promoter-dependent rRNA transcription in Arabidopsis thaliana. Gibberellic acid, abscisic acid, auxin, and ethylene had no detectable effect on rRNA transcription, but kinetin (a cytokinin) stimulated rRNA transcription within 1 h of treatment. Increased steady-state levels of accurately initiated rRNA transcripts, detected by S1 nuclease protection, were paralleled by increased levels of nascent rRNA transcripts in isolated nuclei. Therefore, the primary effect of cytokinin appears to be at the level of transcription initiation rather than rRNA stability. Pol I accounts for approximately 34% of total nuclear transcription in untreated plants and approximately 60% following cytokinin treatment. The specific responsiveness of pol I transcription to kinetin suggests that cytokinins may act as general regulators of protein synthetic capacity and growth status in plant cells.