Genes for histidyl-aspartyl (His-Asp) phosphorelay components (His-containing phosphotransfer proteins, HP, and response regulators, RR) were isolated from Zea mays L. to characterize their function in cytokinin signaling. Six type-A RRs (ZmRR1, ZmRR2, ZmRR4-ZmRR7), 3 type-B RRs (ZmRR8-ZmRR10), and 3 HPs (ZmHP1-ZmHP3) were found in leaves. All type-A RR genes expressed in leaves were up-regulated by exogenous cytokinin. Transient expression of fusion products of the signaling modules with green fluorescent protein in epidermal leaf cells suggested cytosolic and nuclear localizations of ZmHPs, whereas type-B ZmRR8 was restricted to the nucleus. Type-A RRs were localized partly to the cytosol (ZmRR1, ZmRR2, and ZmRR3) and partly to the nucleus (ZmRR4, ZmRR5, and ZmRR6). In the yeast two-hybrid assay, ZmHP1 and ZmHP3 interacted with both cytosolic ZmRR1 and nuclear type-B ZmRRs. In vitro experiments demonstrated that ZmHPs function as a phospho-donor for ZmRRs; turnover rates of the phosphorylated state were tenfold lower in ZmRR8 and ZmRR9 than in ZmRR1 and ZmRR4. These results suggest that the His-Asp phosphorelay signaling pathway might diverge into a cytosolic and a nuclear branch in leaves of maize, and that the biochemical nature of ZmRRs is different in terms of stability of the phosphorylated status.