Thirty-five analogues of Phe-Leu-Glu-Glu-Leu, the pentapeptide sequence 5-9 of bovine prothrombin precursor, were synthesized and assayed as potential substrates or inhibitors of rat liver vitamin K dependent carboxylase. Carboxylation of substrate was determined by measuring the incorporation of carbon-24 labeled bicarbonate into product. Changes in substrate carboxylation produced by changing peptide chain length, amino acid chirality, or the distance separating the peptide chain backbone from the carboxyl group were measured. The data suggest that the carboxylase carboxylates L-glutamic acid residues and does not carboxylate L-aspartic acid, L-homoglutamic acid, glutamine, or D-glutamic acid residues; tri-through pentapeptides are better substrates than mono- or bis(amino acid) derivatives, and hydrophobic groups added to the N-terminus can produce better substrates for the enzyme. None of the synthetic substrates is carboxylated as effectively as the endogenous protein substrates for the enzyme. The effect of structure on additional parameters affecting carboxylation is discussed.