Membrane-binding properties of human and bovine forms of vitamin K- dependent proteins Z, S, and C were characterized. Each of these proteins showed unique properties and interspecies differences that correlated with specific amino acid sequence variations in the amino-terminal 45 residues. Protein Z showed 100-fold slower membrane binding and dissociation kinetics relative to other vitamin K-dependent proteins that have been tested. This property seemed to correlate with an extra γ-carboxyglutamic acid (Gla) residue at position 11 of protein Z. The interspecies difference for protein Z consisted of a higher packing density for the bovine protein on the membrane and a 9-fold slower dissociation rate. Higher affinity correlated with Asp at position 34 of bovine protein Z, where the human protein contains Asn. While both protein S species showed high affinity for the membrane, it was significantly greater for the human protein versus bovine protein S. Again, higher affinity correlated with an Asp (vs Asn) at position 34. Protein C was characterized by binding affinities that were 100-1000-fold lower than the other proteins. Low affinity appeared to be related to loss of Gla-32 (homologous to Gla-33 of protein Z). Interspecies differences of protein C appeared to be related to proline at position 10 (homologous to position 11 of protein Z) of bovine protein C, which produced at least 10- fold lower affinity than the human protein. Comparable substitutions at positions homologous to 11, 33, and 34 of protein Z may also underlie membrane binding behaviors of other vitamin K-dependent proteins. The three- dimensional structure of strontium-prothrombin fragment 1 [Seshadri et al. (1994) Biochemistry 33, 1087] shows that these positions are clustered on the protein surface near strontium-8, another possible candidate for membrane contact. A membrane contact mechanism consisting of an isolated protein- lipid ion pair is proposed. Comparison of naturally occurring vitamin K- dependent proteins has provided possible bases for divergent membrane binding and suggested future approaches to determine biological function.