The tumor suppressor protein p53 is phosphorylated at multiple sites in the amino-terminal transactivation domain and at several sites in the carboxy-terminal region. Phosphorylation appears to modulate its DNA binding activity. Here we demonstrate that phosphorylation of p53 also modulates its transcriptional activity. Okadaic acid treatment of cells resulted in enhanced phosphorylation of p53 and concomitantly in enhanced transactivation of an mdm2 promoter-linked luciferase reporter gene. This effect was cell type specific, however, since transactivation was enhanced in rat and mouse fibroblasts but reduced in the human Saos-2 cell line. Moreover, the effect was dependent on the promoter. In rat cells transcription from the mdm2, waf1 (cip1) and bax gene promoters, and the artificial PG13 promoter was enhanced by okadaic acid treatment whereas that from the cyclin G promoter was reduced. When various phosphorylation site mutants of p53 were tested for transactivation of these promoters, they behaved differently. Amino-terminal mutants exhibited reduced transcriptional activities on mdm2, waf1 and cyclin G promoters but enhanced activities with bax and PG13 promoters. On the other hand, a mutant at the cdk phosphorylation site, A313, showed reduced activity with mdm2 and waf1 promoters but enhanced activity with the cyclin G promoter, and finally, mutant A390 exhibited enhanced activity on waf1 and bax promoters, but reduced activity on the cyclin G promoter. These results suggest that phosphorylation of p53 may have different effects on its transcriptional activity, depending on the cellular environment and the particular response element. Moreover, both, amino- and carboxy-terminal phosphorylation sites seem to be involved in modulating the DNA-binding and the transactivation activities.