The effect of human wild type and mutant p53 proteins on the human multidrug resistance (MDR1) promoter was studied in a p53-negative human cell line. Transient expression of MDR1 promoter-chloramphenicol acetyltransferase reporter gene constructs (MDRCAT) cotransfected with p53 expression vectors was analyzed in H358 lung carcinoma cells. Cotransfection with a wild type p53 expression vector stimulated MDRCAT activity, while cotransfection with mutant p53 expression vectors altered at amino acid positions 181, 252, 258, or 273 failed to stimulate expression. Wild type p53 stimulation of MDRCAT activity was time dependent with maximal expression occurring 24-30 h following transfection and correlating with high p53 protein levels. MDR1 promoter deletion analysis suggested that the sequences involved in wild type p53 stimulation of MDRCAT activity were contained within the region from -39 to +53 relative to the start of transcription at +1. This region contains no TATA or p53 consensus binding sequence but does contain an initiator sequence. Wild type p53 stimulation of MDRCAT expression also occurred in parental and doxorubicin-resistant SW620 colon and parental 2780 ovarian cancer cell lines, indicating that wild type p53-mediated simulation of the MDR1 promoter is not restricted to a single cell line.