Myotonic dystrophy kinase-related Cdc42 binding kinases (MRCKs) are family members most related to the myotonic dystrophy kinase (DMPK), RhoA-binding kinase (ROK), and citron kinase. Two highly conserved members, MRCK␣ and -, have been previously identified and characterized. We now describe a novel isoform, MRCK␥, which is functionally and structurally related to members of this kinase family. We show these kinases to have marked similarities in their genomic organization, substrate phosphorylation, and catalytic autoinhibition. Unlike MRCK␣ and -, which are expressed ubiquitously, MRCK␥ mRNA was only expressed in heart and skeletal muscle. In cultured cells, MRCK␥ showed differential expression with high levels of expression only in certain cell lines. DNA analysis showed that lack of expression is correlated with promoter DNA methylation. We have mapped the methylation sites in the MRCK␥ promoter. Significantly, agents that suppressed DNA methylation caused increases in the expression of the kinase in low-expressing cells, further supporting the notion that promoter DNA methylation plays an important role in the expression of MRCK␥. Analysis of the MRCK␥ promoter has also revealed two proximal Sp1 sites that are essential for transcriptional activity. We conclude that both promoter DNA methylation and Sp1 binding are important regulators for MRCK␥ expression.