The interaction of prothrombin and factor X with membranes of widely varying composition is reported. These protein-membrane interactions require the presence of acidic phospholipid; phosphatidylserine is the most effective from the standpoint of protein-membrane affinity. The maximum protein binding capacity is proportional to the phosphatidylserine content of the membrane up to about 15% and the stoichiometry is 9.2 +/- 1.3 phosphatidylserine residues per prothrombin molecule and 5.2 +/- 1.5 per factor X molecular. The protein apparently causes clustering of the phosphatidylserine residues in these membranes of low phosphatidylserine content. Above about 20% phosphatidylserine, the factor limiting protein-membrane interaction appears to be protein packing density on the membrane surface. The maximum protein binding capacity at 50% phosphatidylserine is 1.2 g of protein per g of phospholipid. Phosphatidic acid is similar to phosphatidylserine in its ability to bind these proteins except for somewhat larger dissociation constants. Phosphatidylethanolamine and phosphatidylglycerol are less effective in all respects for promoting these protein-membrane interactions. Small amounts of phosphatidylserine mixed with these latter phospholipids, however, have a major effect on protein-membrane binding so that the dissociaction constants are more characteristic of membranes of high phosphatidylserine content.