This study was designed to clarify the role of gut microflora in tumorigenesis by a comparison of tumor production between male germ-free and conventional Wistar rats given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 100 microgram/ml in drinking water. Ninety-one % of conventional MNNG-treated rats that died or were killed by Day 314 of the experiment developed tumors in the gastrointestinal tract, whereas only 17% of germ-free treated rats developed such tumors. In addition, large tumors, some 5 cm or more in diameter, were frequently observed in the conventional rats, whereas only small tumors 0.4 to 1.2 cm in diameter were present in the germ-free rats. Furthermore, multiple tumors including double tumors were often found in the conventional rats, while such tumors never appeared in the germ-free rats. The results suggest that gut microflora might exert a promoting influence on tumorigenesis by MNNG in the gastrointestinal tract. The promoting influence of the microflora in conventional rats might not be of a simple nature, since the influence of a variety of factors modified by the micorflora on tumorigenesis by MNNG p.o. is unavoidable.