The concentration-dependent effect of spermine was investigated on the spermine-induced generation of multilayer myotube sheets (MMTS) from mouse embryoid bodies (EBs). During spermine treatment for 24 h, a monolayer cell sheet that had already grown radially from the periphery of an EB was exfoliated. The exfoliation was inhibited by z-VAD.fmk, indicating the occurrence of apoptosis, and inhibited also by aminoguanidine, indicating the involvement of amine oxidase. Following the exfoliation, the cell growth restarted from the fresh periphery of EB in a spermine-free medium and finally formed MMTS. To analyze the contribution of apoptosis to the cell death causing exfoliation, the numbers of apoptotic, necrotic, and 2nd apoptotic cells were counted by staining with Annexin V-Cyanine-3 (AVC3) and 7-aminoactinomycin (7AAC). AVC3-positive, 7AAC-positive, and AVC3/7AAC doubly positive cells were assigned as apoptotic, necrotic, and 2nd necrotic cells, respectively. The relative number of apoptotic and 2nd necrotic cells (N A + N A/7) to the total number of dying cells (N T) was 84 ∼ 94%, which was independent of spermine concentration in the range from 0.1 to 2.0 mM. The MMTS generation rate at the final stage, however, was dependent on the spermine concentration. It was 60 ∼ 80% in the range from 0.1 to 1.5 mM, while it decreased sharply to 1% at 2 mM. This suggests another role of spermine in the MMTS generation in addition to the induction of apoptosis. This 2nd role seems to be inhibited at a spermine concentration higher than a critical limit between 1.5 and 2.0 mM.