We characterize CrRLK1, a novel type of receptor-like kinase (RLK), from the plant Catharanthus roseus (Madagascar periwinkle). The protein (90.2 kDa) deduced from the complete genomic and cDNA sequences is a RLK by predicting a N-terminal signal peptide, a large extracytoplasmic domain, a membrane-spanning hydrophobic region followed by a transfer-stop signal, and a C-terminal cytoplasmic protein kinase with all 11 conserved subdomains. It is a novel RLK type because the predicted extracytoplasmic region shares no similarity with other RLKs. The autophosphorylation was investigated with affinity-purified proteins expressed in Escherichia coli. The activity was higher with Mn 2؉ than with Mg 2؉ and achieved half-maximal rates at 2-2.5 M ATP. The phosphorylation was predominantly on Thr, less on Ser, and not on Tyr. In contrast to other plant RLK, the kinase used an intra-rather than an intermolecular phosphorylation mechanism. After protein cleavage with formic acid, most of the radioactivity was in a 14.1-kDa peptide located at the end of the kinase domain. Mutagenesis of the four Thr residues in this peptide identified Thr-720 in the subdomain XI as important for autophosphorylation and for phosphorylation of ␤-casein. This Thr is conserved in other related kinases, suggesting a subfamily sharing common autophosphorylation mechanisms. Animal receptor protein kinases (RPK)1 located in the membranes play an important role in the perception and transmittance of external signals. All RPK contain an extracellular domain connected by a membrane-spanning amino acid (AA) stretch to the intracellular kinase domain. The proteins can be subdivided into two groups that autophosphorylate on Ser/Thr or on Tyr residues, and oligomerization appears to play an important role in the regulation of enzyme activity (1).Several plant cDNAs and genes for RPK homologs sharing the same basic domain structure have been described. They were called receptor-like kinases (RLK), because the ligands are unknown and their function as receptors has not yet been demonstrated. Several groups are presently distinguished, and the classification is usually based on the sequences in the extracellular domain (reviewed in Ref.2): (a) the S-domain type, with similarities to the S-locus glycoproteins in Brassica; these proteins also contain ten or more cysteines in conserved positions; (b) the leucine-rich repeat type, with 9 -26 Leu-rich repeats; and (c) the epidermal growth factor (EGF)-like type that contains several EGF-like repeats. Very recent data suggest two additional types, with the extracellular region related either to plant defense proteins (3) or to lectins (4), but the investigations have not been extended much beyond the sequences. The physiological functions of plant RLKs are unknown, except for the S-domain type RLKs from Brassica that appear to be involved in the self-incompatibility phenotype (5-7).