A cDNA clone, encoding calcium (Ca2+)-dependent protein kinase (CDPK or CPK), was isolated from tobacco (Nicotiana tabacum). The full-length cDNA of 2360 bp contains an open reading frame for NtCPK4 consisting of 572 amino acid residues. Sequence alignment indicated that NtCPK4 shared high similarities with other CPKs and some CPK-related protein kinases (CRKs). Biochemical analyses showed that NtCPK4 phosphorylated itself and calf thymus histones fraction III-S (histone III-S) in a calcium-dependent manner, and the K0.5 of calcium activation was 0.29 microM or 0.25 microM with histone III-S or syntide-2 as substrates, respectively. The Vmax and Km were 588 nmol min-1 mg-1 and 176 microg ml-1, respectively, when histone III-S was used as substrate, while they were 2415 nmol min-1 mg-1 and 58 microM, respectively, with syntide-2 as substrate. In addition, the phosphorylation of NtCPK4 occurred on threonine residue, as shown by capillary electrophoresis analyses. All of these data demonstrated that NtCPK4 was a serine/threonine protein kinase. NtCPK4 as a low copy gene was expressed in all tested organs including the root, leaf, stem, and flower of tobacco, while its expression was temporally and spatially modulated in both productive and vegetative tissues during tobacco growth and development. NtCPK4 expression was also increased in response to the treatment of gibberellin or NaCl. Our study suggested that NtCPK4 might play vital roles in plant development and responses to environmental stimuli.