Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air-water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2 days whereas it remained stable for B. subtilis 9943 during the 6 days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation.