Many flowering plants possess self-incompatibility (SI) systems to prevent inbreeding. In Brassica, SI recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which encodes both the male determinant (S-locus protein 11 (SP11/SCR)) and the female determinant (S-receptor kinase (SRK)). After self-pollination, an S-haplotype specific interaction between pollen-borne SP11 and its cognate stigmatic SRK receptor induces SI signaling in stigma papilla cells, which results in the rejection of the self-pollen. Previous genetic analysis by our group of a self-compatible mutant revealed the involvement of a membrane-anchored cytoplasmic protein kinase, M-locus protein kinase (MLPK), in SI signaling. The plasma membrane localization of MLPK suggests that it functions in the vicinity of SRK, but the physiological relationship between the two proteins remains unknown. In the present study, we looked for a direct interaction between SRK and MLPK using both genetic and biochemical techniques. Although a conventional yeast two-hybrid system and a split-ubiquitin membrane-based yeast two-hybrid system failed to detect a direct interaction between SRK and MLPK, an in vitro phosphorylation assay indicated that the kinase domain of SRK could efficiently phosphorylate MLPK. These results suggest that MLPK could be a direct target of SRK in Brassica SI signaling.