A lacI-containing transgenic mouse mutation detection system (Big Blue) was used to determine the frequency and spectrum of spontaneous mutation in two rapidly dividing tissues (male germ cells and thymus) and one slowly dividing tissue (brain) at 3 and 10 months of age. By screening 9.4 million lambda plaques, a total of 343 circular mutant plaques were recovered from the three tissues. The mutation frequencies and spectra were determined by sequencing the lacI gene and associated lacZ operator in all samples and correcting for "jackpot" mutations. The mutation frequencies and spectra were similar in all three tissues and there were no age-dependent or gender-dependent changes. When the mutation spectrum in each tissue was compared by utilizing large numbers of independent mutations (average: 75 per tissue), there was evidence for small tissue-specific differences. The spectrum of "jackpot" mutations, which clearly represents in vivo mouse-derived mutations, was similar to that of nonjackpot mutations, providing additional evidence that observed mutations occur in mouse. In the aggregate, the results suggest that there is: (i) a core mutation frequency and spectrum that is modified weakly by tissue-specific metabolism, and (ii) a steady-state level of spontaneous mutation in adult mice reflecting the balance between the accumulation of new mutations and the elimination of mutated cells by either selection against suboptimal cellular function or apoptosis triggered by accumulated DNA damage.