We investigated the effect of various oligosaccharides (OS) on the inhibition of factor IXa by antithrombin (AT) in a purified system. The OS comprised the AT-binding pentasaccharide sequence prolonged by saccharide chains with various lengths and charges. We show that factor IXa inhibition depended on the molecular weight of the OS. Factor IXa was not inhibited by the AT-binding pentasaccharide alone, but was inhibited if it was prolonged by a sulphated dodecasaccharide at the non-reducing end. The overall charge was also important since factor IXa inhibition was negligible if the pentasaccharide was prolonged by a non-sulphated dodecasaccharide. Using compounds containing a non-sulphated spacer, we showed that the central part of the OS was not critical. This study therefore demonstrates that the minimal OS structure necessary for catalysing factor IXa inhibition by AT is close to that required for catalysing thrombin inhibition.