After a single oral dose of 2-nitro[9-14C]fluorene (NF) to germfree and conventional rats, radioactivity with associated mutagenic activity was rapidly excreted in both urine and feces. The mutagenicity excreted from germfree animals exceeded the mutagenicity excreted from conventional animals. Absence of the microflora resulted in lack of nitroreduction and excreted metabolites from germfree rats were mono- or dihydroxylated nitrofluorenes, as assessed by liquid chromatography/mass spectrometry (LC/MS). These hydroxy-NFs were associated with the high direct-acting mutagenicity that was excreted in germfree animals. Only traces of NF were found in urine from germfree rats and no NF was detected in urine from conventional animals. Thus, in contrast to a number of reports on other mutagenic compounds, these results would tend to indicate that the intestinal microflora causes a reduced excretion of mutagenic metabolites of NF.