Infection of the intubated subglottis is felt to be one of the many factors involved in the pathogenesis of acquired cicatricial subglottic stenosis. The precise role of infection is unclear and the microbial flora has not been established. An analysis of subglottic culture material, from 22 intubated pediatric patients undergoing tracheotomy, has been performed to establish the nature of the subglottic microbial flora. Fifty-nine isolates were found, including 19 types of organisms. The number of isolates per patient ranged from one to eight, with an average of 2.7 isolates per patient. The most common isolates in the 22 patients were alpha-hemolytic Streptococcus viridans (17 isolates-77%), Neisseria sp. (6 isolates-27%), Pseudomonas sp. (5 isolates-22%), and coagulase negative Staphylococcus sp. (5 isolates-22%). This data indicates that colonization of the subglottis in intubated pediatric patients is polymicrobial in nature. alpha-Hemolytic Streptococcus viridans and Neisseria sp. were most common, with a shift in cultured flora towards Pseudomonas sp. in patients intubated for more than 10 days. In view of this, antimicrobial therapy may be of benefit in preventing acquired cicatricial subglottic stenosis.