Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu (‘FLEEL’), decarboxylated osteocalcin, decarboxylated fragment 13-29 from descarboxyprothrombin and decarboxylated sperm 4-carboxyglutamic acid-containing (Gla-)protein. The results obtained demonstrated that liver and vessel wall carboxylase may be regarded as isoenzymes with different substrate specificities. The newly discovered enzyme is the first vitamin K-dependent carboxylase which shows an absolute substrate specificity: FLEEL and decarboxylated osteocalcin were good substrates for vessel wall carboxylase, but decarboxylated fragment 13-29 and decarboxylated sperm Gla-protein were not carboxylated at all.