It has been reported that the concentration of free placental growth factor (PIGF) is decreased and that of soluble fms-like tyrosine kinase 1 (sFlt-1) is increased before the onset of preeclampsia. However, no study has determined the reference values for sFlt-1 and free PIGF during pregnancy using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. This longitudinal cohort study was undertaken to address this issue. Serum samples were collected from 148 women at 10, 18, 28, and 37 weeks of gestation. Preeclampsia occurred in 6 women: 4 women who delivered at <37 weeks of gestation, and 2 women who delivered at > or =37 weeks. The average and 90% confidence interval (90% CI) of the serum concentration of both sFIt-1 and free PIGF were determined in a total of 433 specimens from 148 subjects with 1 to 4 collections at 7 to 39 weeks of gestation, and were represented as quadric curves. The mean values (90% CI) of sFlt-1 (pg/ml) at 10, 18, 28, and 37 weeks of gestation were 413 (174-981), 296 (125-704), 413 (174-982), and 1,130 (477-2,690), respectively. The mean values (90% CI) of free PIGF (pg/ml) were 36 (14-89), 206 (83-515), 518 (207-1,290), and 354 (142-884), respectively. We also established the reference values for the ratio of sFlt-1/PIGF. These values may be useful for predicting the subsequent occurrence of preeclampsia.