We tested the hypothesis that nitric oxide (NO) plays an important role in cytokinin signaling. Inhibitors of NO-synthase (NOS), L-NMMA and L-NAME, inhibited the expression of the GUS gene controlled by the cytokinin-responsive ARR5 promoter. However, the inactive analogues D-NMMA and D-NAME had a similar inhibitory activity. NO donors alone did not induce GUS activity and the NO scavenger cPTIO did not prevent the induction of the ARR5 promoter by cytokinin. Northern blot analysis of the P(ARR5)::GUS transgene and the host ARR5 gene revealed that cytokinin-induced transcript accumulation was not altered by NMMA-treatment, indicating that NMMA acts post-transcriptionally. Together the data show that NO has no direct role in eliciting the primary cytokinin response in plants.