Background: Free vascular endothelial growth factor (VEGF) is undetectable in plasma during human pregnancy. However, studies examining pregnant rats have reported both low (8-29 pg/mL) and high (527-1030 pg/mL) free VEGF. These discrepancies cast uncertainty over the use of rat models to study angiogenic factors in pregnancy and preeclampsia. This study investigates methodological factors that may explain these discrepancies. Methods: Plasma VEGF in non-pregnant, day 7 pregnant and day 19 pregnant rats was measured using rat and mouse ELISAs. Results: The rat ELISA detected VEGF in plasma from non-pregnant rats, but not in plasma from day 19 pregnant rats. The mouse ELISA detected higher VEGF concentrations than the rat ELISA in every sample tested. This discrepancy was greater in day 19 pregnant rats (Median: 2,273 vs. 0 pg/mL) than in non-pregnant (97 vs. 20 pg/mL) and day 7 pregnant (66 vs. 2 pg/mL) rats. Recovery of recombinant rat VEGF (rrVEGF) spiked into plasma from non-pregnant and day 7 pregnant rats was high for the rat ELISA (82-105%), but low for the mouse ELISA (17-22%). The rat ELISA did not recover rrVEGF in plasma from day 19 pregnant rats, suggesting that this ELISA measures free VEGF. Conclusions: The use of the rat vs. mouse ELISA likely explains the differences in reported VEGF concentrations in pregnant rats. While the rat ELISA appears to measure free VEGF, plasma concentrations are below the assay sensitivity limit. As most previous studies of pregnant rats used the mouse VEGF ELISA, this data should be interpreted cautiously.