Gardnerella vaginalis is the predominant vaginal microorganism in women with bacterial vaginosis. However, this organism is also frequently isolated from women without signs or symptoms of vaginitis. Earlier studies have not revealed whether certain biotypes of G. vaginalis are more often associated with bacterial vaginosis or are more common in women who acquire bacterial vaginosis. We used a typing scheme based on tests for beta-galactosidase, hippurate hydrolysis, and lipase, using oleate as a substrate. Of 261 strains tested, the distribution of biotypes observed was as follows: 1, 13%; 2, 9%; 3, 5%; 4, 7%; 5, 41%; 6, 15%; and 8, 10%. Biotype 7 was not observed. The distributions of biotypes from women with and without bacterial vaginosis were found to be significantly different, with the lipase-positive biotypes (biotypes 1, 2, 3, and 4) being more predominant in women with vaginosis (41 versus 23%, P = 0.003). Of 40 women with normal vaginal flora at the index visit who remained normal at follow-up, 23 (57%) acquired a new biotype of G. vaginalis. By comparison, 90% of the 30 women who developed bacterial vaginosis acquired a new biotype of G. vaginalis (P = 0.003). Women with bacterial vaginosis at the index visit who were not treated were no more likely than normal women to have a shift in G. vaginalis biotype. However, 86% of the 30 women with bacterial vaginosis who were treated with an antibiotic at the index visit acquired a different biotype (P = 0.04 compared with the value for untreated women) regardless of treatment success. A trend toward the acquisition of a new biotype was observed among women who had contact with a new sexual partner (81 versus 65%, P = 0.15). These data demonstrate that the lipase-positive isolates of G. vaginalis are associated with bacterial vaginosis. Women who acquire bacterial vaginosis are more likely to have a shift in biotype than women who had normal flora at he follow-up, suggesting that the G. vaginalis isolates recovered from women who develop bacterial vaginosis represent newly acquired strains rather than overgrowth of previously colonizing biotypes.