Activated protein C (APC) generation strongly affects sepsis and thrombosis by inhibition of thrombin generation. However, it is unclear if there are age-related differences in effectiveness of protein C (PC). We studied age effects on plasma APC generation ± endothelium. Defibrinated (Ancrod) plasma (from adults or newborns (umbilical cord)) was recalcified with buffer containing tissue factor ± thrombomodulin (TM) on either plastic or endothelium (HUVEC) at 37oC. Timed subsamples of reaction mixture were taken into either heparin-EDTA or FFRCMK-EDTA solutions and analyzed for APC-PC inhibitor (APC-PCI) or APC-α1antitrypsin (APC-α1AT) by ELISAs. Since heparin converts free APC to APC-PCI, the difference in APCPCI measured in heparin-EDTA and FFRCMK-EDTA samples was equal to free active APC. APC-α2macroglobulin (APC-α2M) was measured as remaining chromogenic activity in heparin-EDTA. Free APC, APC-PCI and APC-α1AT were decreased in newborn compared to adult plasma on plastic. However, APC-α2M made up a larger fraction of inhibitor complexes in newborn plasma. On endothelium, significantly more APC, APC-PCI and APC-α1AT were generated in either plasma compared to that on plastic with excess added TM. APC, APC-PCI and APC-α1AT were also reduced and total APC-α2M increased in newborn plasma on HUVEC. Addition of PC to newborn plasma gave APC generation similar to adult plasma. Thus, free APC, APC-PCI and APC-α1AT generation is reduced in newborn compared to adult plasma with or without endothelium, likely due to reduced plasma PC levels. Endothelium enhances APC generation, regardless of plasma type, possibly because of cell surface factors such as TM, phospholipid and endothelial PC receptor.