The clonal composition of 34 benign and malignant sporadic pancreatic endocrine tumours (PETs) of female patients was studied using a sensitive polymerase chain reaction (PCR)-mediated non-isotopic clonality analysis, which is based on the inactivation patterns of polymorphic X-linked genes encoding the androgen receptor (AR) and phosphoglycerate kinase (PGK-1) proteins. Predigestion of DNA with the methylation-sensitive restriction endonuclease Hpa II permitted selective PCR amplification of the methylated (uncleaved) allele. Amplification was successful in 27 of 34 samples. Twenty patient samples were heterozygous for the AR microsatellite region or Bst XI polymorphic site of the PGK-1 gene, permitting analysis of clonality. A monoclonal pattern of X-chromosome inactivation was found in 7 of 20 PETs (35 per cent), since DNA pretreatment with Hpa II blocked amplification of one of the two AR or PGK-1 alleles. One additional tumour exhibited an oligoclonal inactivation pattern and two others a loss of heterozygosity (LOH) at the AR locus, indicative of monoclonality. A random pattern of X-chromosome inactivation and polyclonal cellular composition was observed in the remaining ten PETs (50 per cent). When comparing informative benign and malignant PETs, only 2/7 (29 per cent) benign tumours showed a monoclonal pattern and 8/13 (61 per cent) malignant tumours a monoclonal (5), oligoclonal (1), or LOH (2) pattern. The clonal composition of PETs was not associated with a particular growth pattern, proliferation index or immunohistochemical expression pattern. These findings suggest that PETs might initially represent poly-/oligoclonal neoplastic lesions which are eventually outgrown by a single, more aggressive cell clone with the potential for invasive growth and metastatic spread. 1998 John Wiley & Sons, Ltd.