Following receptor-mediated endocytosis of trimethyllysine-labeled asialofetuin and agalacto-orosomucoid by liver parenchymal and nonparenchymal cells, respectively, the glycoproteins are degraded and the methylated lysine residues released. The free intracellular trimethyllysine is then converted, in addition to 2-N-acetyl-6-N-trimethyllysine, to 4-N-trimethylaminobutyrate, carnitine, and acetylcarnitine. In the presence of 1-amino-D-proline, a vitamin B6 antagonist, the total production from protein-bound trimethyllysine of 4-N-trimethylaminobutyrate, the immediate precursor of carnitine, carnitine, and its acetylated derivative was depressed by as much as 60-80% in perfused rat liver. The decreased synthesis of carnitine was accompanied by an accumulation of 3-hydroxy-6-N-trimethyllysine, and intermediate in the carnitine biosynthetic pathway. The extent of 3-hydroxy-6-N-trimethyllysine accumulation, which was not evident in the absence of added 1-amino-D-proline, depended on the dose of 1-amino-D-proline perfused through the liver. In addition, those effects of 1-amino-D-proline were almost completely reversed by inclusion of pyridoxine in the perfusing medium. These results support the suggestion of a requirement for pyridoxal 5'-phosphate in the biosynthesis of carnitine by the liver.