p53 protein accumulation is triggered following exposure to potentially carcinogenic DNA-damaging agents and other physiological processes. Here we show that although p53 mRNA transcribed from the downstream P(1) transcription start site was the only p53 transcript detected in human cell lines and tumor specimens, p53 transcripts initiated at the upstream P(0) and P(2) start sites were primary in normal human tissues, with P(0)-initiated p53 transcripts comprising approximately 50% of total p53 transcripts. P(1)-initiated p53 mRNA was not detected in most normal human tissues examined. Decreased translational efficiency was observed for mRNAs containing p53 5' untranslated region sequences located between P(0) and P(1) in rabbit reticulocyte lysates and in cell lines; no inhibitory activity was observed for sequences located downstream of the P(1) start site. These data suggest that a transcriptional switch from P(0)-/P(2)- to P(1)-initiated p53 mRNA could be an important mechanism by which cells regulate p53 expression.