Abstract: The micro determination of methotrexate in pharmaceutical preparation and in urine by higher derivative convolution polarography has been reported in this paper. A linear relationship holds between the peak heights and the concentrations in 9.0×10^-8～1.6×10^-6 mol/L range. A comparison with the 1980 British Pharmacopeia method was carried out which showed that convolution polarography has higher sensitivity.Over the pH range of 2～11 in Britton-Robinson buffer, methotrexate is reduced at the DME by way of two polarographic or voltammetric processes. The first, less negative process, is a reversible 2e-2H⁺ reduction of pyrazine ring in the molecule and the second, more negative process, involves an irreversible reduction of pyrimidine ring.