The primary tumors from 15 untreated patients with Burkitt's lymphoma were analysed for abnormalities in the coding region of the MYC gene by single stranded conformational polymorphism (SSCP) analysis followed by DNA sequencing. Fourteen of the 15 tumors had one or more clonal mutations. Forty one mutations were found in the second exon; only one occurred in the third exon. Seven tumors had mutations that clustered in a region spanning amino acids 38-63. Four of these possessed mutations that altered prolines at positions 57 (3), 60 (1), and 63 (1). Seven tumors were mutated in the central portions of the second exon. These occurred at position 95 (2), position 115 (2), position 137 (1), and position 138 (3). Analysis of the published sequences from five lymphoma cell lines and one primary tumor showed a similar clustering of mutations, with all six having mutations in codons between positions 38-63. The regions where mutations occurred have been associated with a variety of properties, including transcriptional activation and cellular transformation. The number and location of mutations showed no correlation with either chromosome 8 or chromosome 14 breakpoints or with the Epstein-Barr virus positivity of the tumors. This unexpected, frequent occurrence of clustered mutations in the second exon of the MYC gene suggests a role for the mutated MYC proteins in the pathogenesis of Burkitt's lymphoma, possibly through altered interactions of this domain with other cellular factors.