Background The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the etiology of these diseases. This study aimed to compare the characteristics of main lactobacillus species between healthy women and women with bacterial vaginosis (BV) by quantitative culture and PCR methods. Main lactobacillus species include L. crispatus, L. gasseri, L. jensenii and L. iners. Methods A total of 150 Women attending Gynecology Outpatient Clinic of Beijing Friendship Hospital, were diagnosed as having BV because three or more of the following criteria were met (standard of Amsel's composite criteria): homogeneous discharge, elevated vaginal pH (pH >4.5), production of amines, and presence of "clue" cells. Those with less than three of the criteria were considered as healthy. Simultaneously, smears were made of vaginal fluid and Gram stained, then were assessed qualitatively as normal (grade I), intermediate (grade II), or consistent with BV (grade III). Gardnerella vaginalis were identified by using Vitek 2 Compact and PCR methods. Lactobacillus species were identified by PCR methods. Gardherella vaginalis and lactobacilli colony counts were determined by calculating the most number of colonies of each species in the appropriate plates (colonies between 10 and 300), corrected by the dilution of the sample in the plates, and multiplied by 10 (to account for plating 100 mu l), in order to get colony forming units per milliliter of vaginal secretion. Results BV was diagnosed in 31% (46/150) patients using the composite criteria, the remainder being regarded as healthy. The majority of patients with BV had a smear assessed as grade III (91%, 42/46) and minority of them had a smear assessed as grade II (9%, 4/46). The majority of healthy women had a smear assessed as grade I (64%, 67/104). Smears assessed as grade II were found (36%, 37/104) among patients diagnosed as healthy, with less than three of the composite criteria. L. crispatus was cultured from 94% of healthy women and 83% of women with BV, with the former colonies count average value of 10(6) and the latter of 10(3). L. gassed, L. iners, and L. jensenii were cultured from 85%, 68% and 43% of healthy women; and 28%, 89% and 44% of BV women, respectively. Conclusions The quantities of four lactobacillus species except L. jensenii had a significant difference between healthy women and women with BV. Our results provide support for the negative association between L. iners and L. gasseri. Although L. crispatus were existent both in healthy and BV positive women's vagina, the numbers of L. crispatus were significantly different for the dominant number in healthy women. Smears of vaginal fluid and Gram stain play an important guiding role in bacteria culture. Chin Med J 2009;122(22):2748-2751