A subtractive hybridization system for isolating differentiation associated gene from human cancer cell line is developed. By using the strategy of differentiation-inducing in combina tion with cDNA-cDNA subtractive hybridization, a subtracted cDNA library from human lung adenocarcinoma cell line before and after treatment with all-trans retinoic acid(RA) was established. Three cDNAs response to RA treatment were obtained by screening of the subtracted cDNA library. The results of sequencing and function analysis show that three cDNAs are novel cDNAs of differentiation associated genes in human lung adenocarcinoma cell line. This system is also suitable for isolating cDNAs representing deleted or overexpressed gene in human cells.