Endocytosis and the lysosome system have beenstudied in rat myotubes differentiated in vitro. Horseradishperoxidase was used as marker for endocytosis and wasfound to accumulate unevenly in the myotubes. Small segmentsof myotubes display very high endocytotic activity.Similar segments contained numerous lysosomes, as seenby the accumulation of neutral red or histochemical stainingfor acid phosphatase. The segments also contained accumulationsof acetylcholine receptors as determined by bindingof tetramethyl rhodamine-labelled c~-bungarotoxin. Unstainedsegments in living cultures could be recognized byphase-contrast microscopy since they often appeared somewhatdilated and were not as well spread on the culturesurface as the main parts of the myotubes. Ultrastructurally,the segments contained an intensely proliferating tubularsystem in communication with the extracellular space,which therefore probably represents the developing transversetubular system. The segments also contained endocytosedmarker within large phagosomes. Contractile filamentsoccurred in the segments but were frequently lesswell-organized than in other parts of the myotubes. Thedescribed characteristics of the segments in rat myotubesdifferentiated in vitro bear resemblance to some of the characteristicsof the denervated endplate region of adult muscle.