Liquid chromatography and tandem mass spectrometry without prior fractionation (shotgun proteomics) were used to analyze vaginal fluid from patients admitted for signs and symptoms of preterm labor. The patients had an average age of 26.3 +/- 5.9 years, a gestational age of 30.5 +/- 2.5 weeks, and a median cervical dilation of 1 cm (range, 0-6 cm). None of the patients exhibited signs of vaginal infection at the time of enrollment. Shotgun proteomics yielded reproducible identifications (R = 0.973) of more than 40 proteins in vaginal fluid samples, such as plasma proteins, epithelial structural proteins, and several immunoregulatory proteins, including some that were previously linked to intra-amniotic infection. This initial characterization of the vaginal fluid proteome using a nonbiased, high-throughput technique yields reproducible results in late pregnancy. The presence of host defense proteins in vaginal fluid suggests that this technique may be useful for future study of inflammation-related preterm birth.