We previously showed that NtCDPK1, a tobacco cal-cium-dependent protein kinase, interacts with and phosphorylates the Rpn3 regulatory subunit of the 26S proteasome, and that both NtCDPK1 and Rpn3 are mainly expressed in rapidly proliferating tissues, in-cluding shoot and root meristem. In this study, we ex-amined NtCDPK1 expression in roots using GUS ex-pression in transgenic Arabidopsis plants, and investi-gated its function in root development by generating transgenic tobacco plants carrying a sense NtCDPK1 transgene. GUS activity was first detected in roots two days after sowing. In later stages, strong GUS expres-sion was detected in the root meristem and elongation zone, as well as the initiation sites and branch points of lateral roots. Transgenic tobacco plants in which NtCDPK1 expression was suppressed were smaller, and their root development was abnormal, with reduced lateral root formation and less elongation. These re-sults suggest that NtCDPK1 plays a role in a signaling pathway regulating root development in tobacco.