Previous findings that the intermediate filament nestin is expressed in immature skeletal muscle cells prompted us to compare the staining patterns of nestin and desmin in rhabdomyosarcomas (RMSs) and in other small cell tumors of infancy. We found that nestin immunoreactivity was present in all of 29 examined typical RMSs, which also expressed desmin. Two undifferentiated tumors, primarily suspected to be RMSs, expressed nestin, but not desmin. One of these nestin-positive, desmin-negative tumors was positive for the expression of the myogenic regulatory gene MyoD and is considered to represent an undifferentiated RMS. The other, a paratesticular tumor, did not contain transcripts for MyoD, and most likely does not represent a RMS. In several RMSs and nonmuscle tumors, a z-disc-associated nestin immunoreactivity occurred as a paramalignant phenomenon in cross-striated muscle fibers adjacent to the tumor cells. Our findings indicate that nestin, although present also in tumors of the central and peripheral nervous systems, as well as in endothelial cells and in some muscle cells adjacent to tumors, is a useful complementary marker for RMS, particularly in very undifferentiated desmin-negative tumors.