In this report we define the genes of two-component regulatory systems in rice through a comprehensive computational analysis of rice (Oryza sativa L.) genome sequence databases. Thirty-seven genes were identified, including 5 HKs (cytokinin-response histidine protein kinase) (OsHK1-4, OsHKL1), 5 HPs (histidine phosphotransfer proteins) (OsHP1-5), 15 type-A RRs (response regulators) (OsRR1-15), 7 type B RR genes (OsRR16-22), and 5 predicted pseudo-response regulators (OsPRR1-5). Protein motif organization, gene structure, phylogenetic analysis, chromosomal location, and comparative analysis between rice, maize, and Arabidopsis are described. Full-length cDNA clones of each gene were isolated from rice. Heterologous expression of each of the OsHKs in yeast mutants conferred histidine kinase function in a cytokinin-dependent manner. Nonconserved regions of individual cDNAs were used as probes in expression profiling experiments. This work provides a foundation for future functional dissection of the rice cytokinin two-component signaling pathway.