A panel of 18 Burkitt's lymphoma (BL) and nine other cell lines was examined for mutations in the N-terminal transactivation domain of c-Myc. Mutations leading to exchange of amino acids were detected in 13 BL but in none of the control cell lines. Mutations in c-Myc clustered between amino acid positions 57 and 62. Thr-58 and Ser-62 are known phosphorylation sites of c-Myc in vivo. BL cell lines derived from the same tumour revealed different mutations. Mutant cDNAs of the BL cell line Raji differed at 14 positions indicating ongoing mutation of the translocated c-myc during long-term propagation in cell culture. The effect of mutations on transactivation by c-Myc was tested by expression of GAL4/c-Myc fusion proteins in the BL cell line Raji. Mutants with an amino acid exchange at positions 58 or 60 transactivated a reporter gene two- to fivefold weaker than wildtype c-Myc. Thr-58 and Ser-62 were replaced by aspartic acid to mimic constitutively phosphorylated forms of c-Myc. These mutants transactivated two- to three-fold weaker than wildtype c-Myc indicating that a negative charge at positions 58 and/or 62 per se does not enhance transactivation. We propose that mutations in the N-terminal domain of c-Myc correlate with reduced transactivation and provide a growth advantage for BL cells.