Implication of His-to-Asp and/or Asp-to-His (His-Asp) phosphorelay has been recently reported in signal transduction pathways initiated by ethylene and cytokinin. These signaling systems are generally composed of sensor His-protein kinases, His-containing phosphotransfer (HPt) domains, and response regulator domains. In this study, we isolated maize cDNAs, designated as ZmRR2 and ZmHP2, which encode a response regulator domain and HPt domain, respectively, and we identified their His-to-Asp phosphotransfer activity in vitro. The putative translated product of ZmRR2 was highly similar to that of ZmRR1 (78% identity), a maize response regulator homologue. The putative translated product of ZmHP2 showed similarity to that of HPt domains from Arabidopsis thaliana (AHP1-AHP3: 44 to 47% identity) and Saccharomyces cerevisiae (Ypdlp: 24% identity). In vitro experiments demonstrated that the putative signaling factors can transfer the phosphoryl group from His-80 of ZmHP2 to Asp-90 of ZmRRs. Treating detached leaves with t-zeatin or supplying inorganic nitrogen to the whole plant induced the accumulation of ZmRR1 and ZmRR2 transcripts. On the other hand, the steady-state transcript level of ZmHP2 was not affected by cytokinin or inorganic nitrogen sources. These results indicate that His-Asp phosphotransfer may be involved in the transduction of nitrogen signals mediated by cytokinin, and that multiple response regulators participate in the signaling pathways.