The full‐length cDNA encoding the entire open reading frame (ORF) of rat myotubularin (rMTM) was isolated from a rat testis expression library by PCR. Among the three ∼2.9‐kb cDNAs that were sequenced, one clone was different from the other two clones. It contained seven extra amino acids of FVVLNLQ; this short stretch of extra sequence was found between Gln⁴²¹ and Phe⁴²² within the SET (Suvar3–9, Enhancer‐of‐zeste, Trithorax) interacting domain (SID) of rMTM. The rMTM ORF had 1,713 bp encoding for a 571 amino acid polypeptide and a calculated molecular weight of 65.8 kDa. A comparison between its deduced amino acid sequence and the GenBank database using BLAST revealed a 53.1% identity with human myotubularin protein (hMTM1), which is a member of the protein tyrosine phosphatase (PTP) family associated with X‐linked myotubular myopathy. A 22 amino acid peptide NH₂‐TKVNERYELCDTYPALLAVPAN was synthesized based on the deduced amino acid sequence of rMTM and used for antibody production. By using immunoblot analysis, a 66‐kDa protein was indeed detected in both Sertoli and germ‐cell cytosols. rMTM mRNA was found in various tissues but was predominantly expressed in the testis, ovary, and skeletal muscle. Sertoli cell rMTM expression was stimulated by germ cells and enhanced when inter‐Sertoli junctions were being assembled in vitro. A drastic reduction in testicular rMTM steady‐state mRNA level correlated with the depletion of germ cells from the testis in vivo following either glycerol or lonidamine treatment. These results indicate that rMTM is a rat homologue of hMTM1 that may be a useful marker in monitoring the events of cell–cell interactions in the testis. J. Cell. Physiol. 185:366–385, 2000. © 2000 Wiley‐Liss, Inc.