Expression of the human papillomavirus 16 E6 oncogene interferes with several vital cellular processes, including the p53-dependent response to DNA damage. To assess the influence of E6 on the early response to DNA damage, we analyzed gene expression following mitomycin C-induced genotoxic stress in human E6-expressing U2OS cells (U2OSE64b) as well as in p53-expressing control cells (U2OSE6AS) by comparative global expression profiling. As expected, genes involved in p53-dependent pathways were activated in p53-expressing cells. In the U2OSE64b cells, however, a largely nonoverlapping group of genes was identified, including two splicing factors of the SR family. Immunoblot analysis revealed increased expression of several SR proteins during the early response to DNA damage, which was accompanied by activation of alternative splicing activity. Disruption of splicing activity by treatment with small interfering RNA directed against splicing factor SRp55 resulted in the increased viability of p53-deficient cells following DNA damage. To determine whether the transient activation of splicing activity was due to E6-mediated degradation of p53, or was due to some other activity of E6, we compared the early response of the p53 wild-type and p53-/- isogenic HCT116 cell lines, and found that the increase in splicing activity was observed only in the absence of p53. Finally, both the U2OSE64b and the p53-/- cells showed altered splicing patterns for the CD44 receptor. Together, these data show that cells lacking p53 can activate alternative splicing following DNA damage.