The possible role of the indigenous intestinal microflora in the toxicoinfection of human infant botulism was studied with adult germfree mice. Intraintestinal botulinum monoassociation was consistently produced when mice were fed 10 C. botulinum type A spores. Control germfree mice became enterically infected when placed in the same isolator with, but separated from, animals that had been fed spores. When transferred into a room holding a colony of normal mice, the highly susceptible gnotobiotes became resistant to challenge of 10(5) spores after about 3 days of the conventionalizing exposure. The findings are interpreted as evidence that enteric botulinum infection occurs in human infants whose intestinal tract has not yet been colonized by bacteria which are indigenous to adults and prevent growth of C. botulinum. Intestinal monoassociation could not be developed in germfree infant mice younger than 7 days.