We first purified native full-length myotonin protein kinase (MtPK), the protein product of the myotonic dystrophy gene, from rat skeletal muscle and demonstrated its kinase activity. This 70 kDa MtPK is localized in the sarcoplasmic reticulum fraction, whereas the previously reported 55 kDa protein is observed in nuclear extracts and the sarcoplasmic reticulum membrane. The purified MtPK shows an autophosphorylation activity. We also demonstrated the colocalization of a 43 kDa MtPK fragment in the sarcoplasmic reticulum with native MtPK. Based on the cDNA sequence, human MtPK was previously reported to have two amino acid sequence variations at the C-terminus, one LTAVWRRPGAARAP (RAP type) and the other SGAAQEPPALPEP (PEP type). The MtPK and 43 kDa fragment purified here appear to be almost entirely RAP type (approx. 0.54 μg/mg sarcoplasmic reticulum protein). From these results, we conclude that MtPK is a sarcoplasmic reticulum membrane protein that may be involved in the signal-transduction pathway causing myotonia.