By using immunohistology combined with immunoblotting, cell culture, and RT-PCR, we show that the intermediate filament protein nestin is transiently expressed in the midembryonic mouse heart. Monoclonal antibody (MAb) Rat-401, known to react with nestin in neural and skeletal muscle cells, was also found to react with ventricular and atrial cells throughout the mouse heart from embryonic day 9 (E9) through E10.5. Both before (E8.5) and after (E11-adult) this brief period, staining with Rat-401 was absent from atrial and ventricular myocytes. To evaluate the specificity of staining with MAb Rat-401 in the heart, we used immunoblotting, cell culture, and RT-PCR to verify that the authentic nestin protein and mRNA were expressed in cardiomyocytes of the E10 mouse. Nestin expression is the first molecular marker for this distinct midembryonic period of heart development.