In a longitudinal cohort study of 1,248 nonpregnant young women recruited from three Pittsburgh, Pennsylvania, health clinics in 1998-2000, the authors investigated risk factors associated with vaginal acquisition of group B Streptococcus (GBS). Rectal and vaginal swabs for GBS culture and demographic and behavioral interview data were obtained from the women at enrollment and at three 4-month intervals. Cox proportional hazards models were used to evaluate risk factors for GBS acquisition among the 1,089 women with follow-up data. At enrollment, 365 (29.2%) of the 1,248 study participants were vaginally colonized with GBS. Of 767 women who were GBS-negative at enrollment, 344 (44.9%) acquired vaginal GBS colonization during follow-up. The following factors were independently associated with vaginal acquisition of GBS at the 0.05 significance level: African-American race (hazard ratio (HR) = 1.5, 95% confidence interval (CI): 1.2, 1.9), having multiple sex partners during the past 4 months (HR = 1.7, 95% CI: 1.1, 2.5), having frequent sexual intercourse during the past 4 months (HR = 1.5, 95% CI: 1.1, 2.2), and having sexual intercourse within the 5 days prior to the follow-up visit (HR = 1.6, 95% CI: 1.3, 2.0). These results show that sexual activity is an important risk factor for vaginal acquisition of GBS.