BACKGROUND: Several placental microRNAs (miRNAs) have been identified as pregnancy-associated molecules with the potential for use in estimating the condition of the placenta. Our understanding of these novel molecules is still limited, however. The aim of this study was to isolate and characterize pregnancy-associated miRNAs in maternal plasma. METHODS: By microarray-based screening of 723 human miRNAs, we selected miRNAs that exhibited signal intensities >100 times higher in placental tissues than in the corresponding whole blood samples. Subsequent quantitative real-time reverse-transcription PCR revealed miRNAs produced predominantly in the placenta that showed significantly decreased concentrations in maternal plasma after delivery. These miRNAs were identified as pregnancy-associated miRNAs. RESULTS: We selected 82 miRNAs produced predominantly in the placenta and identified 24 as pregnancy-associated miRNAs. The genes encoding these miRNAs included 16 that are clustered on 19q13.42 and 5 clustered on 14q32. As the pregnancy progressed into the third trimester, the plasma concentrations of cell-free chromosome 19–derived miRNAs (has-miR-515-3p, has-miR-517a, has-miR-517c, has-miR-518b, and has-miR-526b) increased significantly (P = 0.0284, 0.0069, 0.0125, 0.0284, and 0.0093, respectively, Wilcoxon signed rank test), whereas that of cell-free has-miR-323-3p on chromosome 14q32.31 showed no change (P = 0.2026). CONCLUSIONS: In addition to the known pregnancy-associated miRNAs, we identified new pregnancy-associated miRNAs with our microarray-based approach. Most of the genes encoding these miRNAs were clustered on 19q13.42 or 14q32, which are critical regions for placental and embryonic development. These new pregnancy-associated miRNAs may be useful molecular markers for monitoring pregnancy-associated diseases.