Two metabolites formed by rat caecal contents or human E. coli (W3110 strain) incubated with 4, 6-dinitro-2-sec-butylphenol (DNBP), one of the agricultural chemicals of dinitrophenol homologous series, were detected by thin-layer chromatography. These metabolites, 6-amino-4-nitro-2-sec-butyl-phenol (6A4NBP) and 6-acetylamino-4-nitro-2-sec-butylphenol (6AA4NBP), were identified by comparing their infrared and mass spectral data with those of authentic samples. The amounts of 6A4NBP and 6AA4NBP which were formed after 48 h incubation were determined by densitometric analysis to be about 63 and 7% for rat caecal contents ; 1.7 and 0.9% for human E. coli, respectively. The results of this investigation indicated that a primary metabolic reaction of DNBP in the rat caecal microflora was the reduction of a nitro group to an amino group, and that N-acetylation of the amino group occurred as the secondary reaction. In addition, densitometric analysis adopted in the present study was confirmed to be very effective for the determination of DNBP and its metabolites.