The Ca²⁺-dependent protein kinases (CDPKs) and abscisic acid (ABA) are known to be involved in low-temperature stress response. The focus of this study was to characterize the 45 kDa protein kinase identified in the crude extract of rice (Oryza sativa L.) seedling roots in response to cold (5°C) stress. The activity of the 45 kDa protein kinase decreased at low temperature as evident by an in-gel kinase assay using histone III-S as a substrate. Also, the Ca²⁺-dependent activity of this protein kinase was suppressed by cold in the membrane fractions of the root. A general protein kinase inhibitor and Ca²⁺ chelator inhibited the activity of the 45 kDa protein kinase, suggesting that it was a plant CDPK. The 45 kDa CDPK identified was found to be independent of photosynthetic tissues such as the leaf and leaf sheath of rice seedlings, supporting a direct sensing mechanism in the roots of rice seedlings to cold stress. The suppressed activity of the 45 kDa CDPK was reverted by supplementing with 5 μM ABA under cold stress. The 45 kDa CDPK activity was stronger in the cold-tolerant variety of the 4 types tested than it was in the cold-sensitive one. These results suggest the involvement of endogenous ABA in regulating the activity of the 45 kDa CDPK in response to cold stress.