Undomesticated strains of Bacillus subtilis can form pellicle biofilms in standing culture. Pellicle formation is initiated by repression of flagellar genes and activation of the eps and yqxM operons, which are involved in biofilm-matrix synthesis. SinI is thought to induce the eps and yqxM operons by antagonizing their repressor SinR. Here, we show that mutations in late-flagellar genes prevent pellicle formation at an initiation step. These mutations reduce the activity of SlrR/SlrA. SlrR (formerly Slr) and SlrA are homologues of SinR and SinI respectively, and SlrR/SlrA represses sigma(D)-dependent flagellar genes and activate the eps and yqxM operons. Contrary to previous reports, a sinI mutation does not prevent pellicle formation in B. subtilis strain ATCC 6051. ATCC 6051 has a frameshift mutation in the ywcC gene, which encodes a TetR-type transcriptional repressor. The ywcC mutation depresses slrA transcription, thereby increasing SlrR/SlrA activity. In the ywcC mutant, SlrR/SlrA rather than SinI activates the eps and yqxM operons by antagonizing SinR. The roles of SlrR/SlrA and flagella in the initiation of pellicle formation are discussed.