Field cancerisation of the aerodigestive tract is caused by chronic exposure to alcohol and tobacco, but the nature of the genetic alterations preceding overt malignancy is unknown. To identify potential field changes we have used a functional assay which tests the transcriptional competence of human p53 expressed in yeast. To increase the sensitivity and reliability of the technique for samples containing under 20% mutant p53, the 5′ and 3′-ends of the p53 cDNA were examined separately. With this split form of the assay the tissue p53 mRNA acts as its own control for RNA quality. Mutations were detected in 87%(46/53) of tumours, reflecting the high sensitivity of the technique. Multiple biopsies of histologically normal tissue from the upper aero-digestive tract were tested and clonal p53 mutations were identified in 76%(38/50) of biopsies from patients presenting with multiple tumours compared with 32%(38/117) of biopsies from patients presenting with single tumours (P< 0.000001). All patients (16/16) presenting with multiple tumours had at least one positive biopsy, compared with only 53%(19/36) of patients presenting with single tumours (P< 0.001). This defines expansion of multiple clones of mutant p53-containing cells as an important biological mechanism of field cancerisation, and provides a means to identify patients likely to benefit from intensive screening for the development of new head and neck tumours.