Tärnberg M, Jakobsson T, Jonasson J, Forsum U. Identification of randomly selected colonies of lactobacilli from normal vaginal fluid by pyrosequencing of the 16S rDNA variable V1 and V3 regions. APMIS 2002;110:802–10. The present study aimed to characterize lactobacilli in vaginal fluid from 23 adult healthy women by using high-throughput DNA sequencing for identification of a large number of randomly selected colonies appearing on Rogosa and blood agar. The typing method was based on broad-range PCR of 16S rRNA gene variable regions V1 and V3, pyrosequencing, and classification of the fragments by alignment with NCBI-catalogued sequences and type strain sequences. Four major groups of sequences were found among the 402 isolates clearly corresponding to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii when compared to the sequences obtained for type strains. Our results indicate that pyrosequencing of 16S rRNA gene fragments as used here is a fast and reliable method well suited for identification to the species level, even within the Lactobacillus acidophilus complex.