Dexamethasone (DEX)-mediated inhibition of drug-induced, but not CD95 ligand-induced, apoptosis in malignant glioma cells correlates with wild-type p53 status. Here, we examined mechanisms underlying DEX-mediated protection from apoptosis. DEX did not induce p53 expression in two p53 wild-type cell lines (U87MG, LN-229) and did not alter drug-induced p53 accumulation. Forced expression of temperature-sensitive p53val 135 in mutant conformation failed to prevent accumulation of endogenous wild-type p53 but acted in a transdominant negative manner to inhibit p53-mediated, camptothecin-induced p21 WAF1/CIP1 expression. p53val 135-transfected cells retained responsiveness to DEX at restrictive temperature, suggesting that p53 activity is not required for cytoprotection. Forced expression of wild-type p53val 135 abrogated the protective effect of DEX, suggesting redundant cytoprotective effects of DEX and p53. Indeed, DEX induced moderate accumulation of p21 WAF1/CIP1 in U87MG, LN-229 and p53 mutant LN-18 cells, but not in p53 mutant LN-308 or T98G cells. LN-18 is also the p53 mutant cell line with the best cytoprotective response to DEX. p21 WAF1/CIP1 accumulation occurred in the absence of changes in p21 WAF1/CIP1 mRNA expression. Wild-type p53 was not required for this DEX effect since DEX induced p21 WAF1/CIP1 accumulation in p53val 135-transfected LN-229 cells, too. DEX failed to induce p21 WAF1/CIP1 expression or cytoprotection in untransformed rat astrocytes. The same lack of modulation of p21 WAF1/CIP1 expression and drug toxicity was observed in p21+/+, p21+/− and p21−/− human colon carcinoma cells. Paradoxically, while only p21+/+ and p21+/− mouse embryonic fibroblasts showed enhance p21 WAF1/CIP1 levels after exposure to DEX, only p21−/− fibroblasts were protected from drug toxicity by DEX. The present study links DEX-mediated protection from cancer chemotherapy to a p53-independent pathway of regulating p21 WAF1/CIP1 expression in glioma cells but this effect appears to cell type-specific.