The nuclear protein prothymosin-α (ProTα), which lacks a signal peptide sequence, is released from neurons and astrocytes on ischemic stress and exerts a unique form of neuroprotection through an anti-necrotic mechanism. Ischemic stress-induced ProTα release is initiated by a nuclear release, followed by extracellular release in a non-vesicular manner, in C6 glioma cells. These processes are caused by ATP loss and elevated Ca²(+), respectively. S100A13, a Ca²(+)-binding protein, was identified to be a major protein co-released with ProTα in an immunoprecipitation assay. The Ca²(+)-dependent interaction between ProTα and S100A13 was found to require the C-terminal peptide sequences of both proteins. In C6 glioma cells expressing a Δ88-98 mutant of S100A13, serum deprivation caused the release of S100A13 mutant, but not of ProTα. When cells were administered apoptogenic compounds, ProTα was cleaved by caspase-3 to generate a C-terminal peptide-deficient fragment, which lacks the nuclear localization signal (NLS). However, there was no extracellular release of ProTα. All these results suggest that necrosis-inducing stress induces an extacellular release of ProTα in a non-vesicular manner, whereas apoptosis-inducing stress does not, owing to the loss of its interaction with S100A13, a cargo molecule for extracellular release.