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Objective: To compare the efficacy of three culture media to detect vaginal and rectal colonization by group B streptococci (GBS) in pregnant women., Methods: We processed 1334 samples from 861 pregnant women: in 388 patients only vaginal swab was cultured (Group A) and in 473 vaginal and rectal swabs were cultured (Group B). Samples were inoculated on blood agar with colistin-nalidixic acid (BA-CNA), on plates with Granada medium, and on Todd-Hewitt broth (THB) with amikacin followed by subculture in BA-CNA., Results: GBS was isolated in 181 samples (13.6%): 114 vaginal swabs (13.2%) and 67 rectal swabs (14.2%). Among the positive samples, GBS was isolated on BA-CNA in 60.5% of vaginal swabs and in 59.7% of rectal swabs, on Granada medium in 80.7% of vaginal swabs and in 91% of rectal swabs, and on THB in 97.4% of vaginal swabs and in 97% of rectal swabs.We detected 130 GBS carriers, 54 (13.9%) in Group A and 76 (16.1%) in Group B. The percent of carriers detected in groups A and B, respectively, was 59.3% and 75% with BA-CNA, 77.8% and 93.4% with Granada medium, and 96.3% and 97.4% with THB., Conclusions: THB was the most reliable medium for the detection of GBS. Use of Granada medium allows fast detection of about 87% of carriers. The combination of THB and an initial plate of Granada medium or BA-CNA allows detection of more than 99% of GBS carriers.