Molecularly imprinted microspheres were synthesised using the antitumor drug piritrexim (PTX) as a template molecule by aqueous microsuspension polymerisation and were used as a high-performance liquid chromatographic stationary phase. The molecularly imprinted column exhibited strong retention behaviour to the template molecule. The influences of pH of the buffer and the ratio of methanol to buffer on the retention behaviour were investigated in detail. Results indicated that the baseline separation of PTX, trimetrexate (TMX), trimethoprim (TMP) and sulfamethazine (SMZ) was achieved when the pH value of the acetate buffer was above pH 3.5 and the ratio of methanol to the buffer was 6:4 (v/v). A gradient elution programme was employed to enhance the separation, which led to an improvement in sensitivity and a reduction in determination errors. The method developed was used to analyse urine samples supplemented with PTX. The recoveries of 5 microg mL(-1) PTX in the urine sample were 99.1+/-3.0% and 93.3+/-2.8% at the beginning and 24 h later, respectively.