The RNA helicase p68 is a potent co-activator of p53-dependent transcription in response to DNA damage. Previous independent studies have indicated that p68 and the Δ133p53 isoforms, which modulate the function of full-length p53, are aberrantly expressed in breast cancers. Here we identify a striking inverse association of p68 and Δ133p53 expression in primary breast cancers. Consistent with these findings, small interfering RNA depletion of p68 in cell lines results in a p53-dependant increase of Δ133p53 in response to DNA damage, suggesting that increased Δ133p53 expression could result from downregulation of p68 and provide a potential mechanistic explanation for our observations in breast cancer. Δ133p53α, which has been shown to negatively regulate the function of full-length p53, reciprocally inhibits the ability of p68 to stimulate p53-dependent transcription from the p21 promoter, suggesting that Δ133p53α may be competing with p68 to regulate p53 function. This hypothesis is underscored by our observations that p68 interacts with the C-terminal domain of p53, co-immunoprecipitates 133p53α from cell extracts and interacts only with p53 molecules that are able to form tetramers. These data suggest that p68, p53 and 133p53α may form part of a complex feedback mechanism to regulate the expression of Δ133p53, with consequent modification of p53-mediated transcription, and may modulate the function of p53 in breast and other cancers that harbour wild-type p53.