L-Glutamine (Gln) starvation rapidly triggers apoptosis in Sp2/0-Ag14 (Sp2/0) murine hybridoma cells. Here, we report on the role played by the stress-activated kinase p38 mitogen-activated protein kinase (MAPK) in this process. p38 activation was detected 2 h after Gln withdrawal and, although treatment with the p38 inhibitor SB203580 did not prevent caspase activation in Gln-starved cells, it reduced the occurrence of both nuclear condensation/fragmentation and apoptotic body formation. Similarly, transfection of Sp2/0 cells with a dominant negative p38 MAPK reduced the incidence of nuclear pyknosis and apoptotic body formation following 2 h of Gln starvation. Gln withdrawal-induced apoptosis was blocked by the overexpression of the anti-apoptotic protein Bcl-xL or by the caspase inhibitor Z-VAD-fmk. Interestingly, Bcl-xL expression inhibited p38 activation, but Z-VAD-fmk treatment did not, indicating that activation of this MAPK occurs downstream of mitochondrial dysfunction and is independent of caspases. Moreover, the anti-oxidant N-acetyl-l-cysteine prevented p38 phosphorylation, showing that p38 activation is triggered by an oxidative stress. Altogether, our findings indicate that p38 MAPK does not contribute to the induction of apoptosis in Gln-starved Sp2/0 cells. Rather, Gln withdrawal leads to mitochondrial dysfunction, causing an oxidative stress and p38 activation, the latter contributing to the formation of late morphological features of apoptotic Sp2/0 cells.