A method for studying steady-state kinetics of nucleotide analogues incorporation into DNA strand by non-gel CE (NGCE) with LIF was developed. Nucleoside analogue is a kind of antiviral agent used to inhibit viral replication in infected cells, especially HIV. Steady-state parameter K-m for nucleotide analogues is determined to imply the relationship between nucleoside analogues and the enzyme in the DNA chain elongation and predict the antiviral efficacy in vivo. Samples were prepared by single nucleotide incorporation assays catalyzed by Taq DNA polymerase at 58 degrees C and HIV reverse transcriptase (RT) at 37 degrees C, and then were separated using NGCE under optimized conditions: 25 mmol/L Tris-boric-EDTA buffer (pH 8.0) with 7 mmol/L urea in the presence of 20%w/v PEG 35000 at 30 degrees C and -20 kV. K-m(dTTP), K-m(d4TTP) and K-m(AZTTP) were measured by NGCE for the first time and their values for Taq DNA polymerase were 0.29 +/- 0.04, 32.1 +/- 3.3 and 74.5 +/- 6.6 mu mol/L, respectively. For HIV RT, the values were 0.15 +/- 0.05, 0.31 +/- 0.03 and 0.17 +/- 0.03 mu mol/L, respectively. The trend of data for HIV RT measured by NGCE was consistent with that measured by PAGE. The reported method by NGCE for the K-m determination was powerful, sensitive and fast, and required less amounts of reagents compared with PAGE. It be employed as a reliable alternative method and further applied in other relative studies of nucleoside analogue substrates and DNA polymerases or RTs.