A phosphonate analogue of pyridoxal 5'-phosphate containing a 5'-phosphonomethyl group and its monoethyl and diethyl esters have been prepared. Except for the diethyl ester, the compounds appear to bind into the active site of aspartate aminotransferase. However, they lack detectable catalytic activity with this enzyme and with glutamate decarboxylase of Escherichia coli. The phosphonomethyl analogue bound to aspartate aminotransferase does react slowly with substrates, as determined by spectrophotometric observations; the monomethyl ester reacts about 20 times less rapidly. Because of the stability of the phosphonate linkage, these compounds may be useful as modifying reagents for various proteins.