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The aim of this study was to explore the effect of different conditions on two-dimensional gel electrophoresis of proteins from human acute promyelocytic leukemia cell line NB4. The 24 cm pH 3-10 linear immobilized pH gradient (IPG) strips were chosen, the isoelectric focusing was carried out by using IPGphor. Then, the second-dimensional SDS-PAGE was performed. After silver staining, the gel was analyzed by ImageMaster 2D Elite. The results showed that low ion intensity sample washing buffer improved the performance of isoelectric focusing. The lysis buffer containing 7 mol/L urea and 40 mmol/L DTT could solubilize the most proteins from NB4 cell line. The rehydration solution containing thiourea and urea increased the low molecular weight protein points to be resolved in the area of basic end. The reasonable sample load and Volt/hour of NB4 were about 100 micro g and 63 200 V/h for the 24 cm pH 3-10 IPG strips. It is concluded that the proteins from NB4 and similar cell line are complicated and affected by many factors, so that, it is very important to select the right methods for sample preparation and the conditions of two-dimensional gel electrophoresis.