In prokaryotes, a number of endogenous antisense RNAs have been detected and found to exert various biological functions. In eukaryotes antisense RNAs have been found; however, a lack of experimental methodologies that permit the identification of overlapping transcripts in cells presents a barrier to a more systematic identification of antisense RNA. Here we have developed an experimental strategy that allows systematic identification of endogenous mRNAs with long complementary regions to other transcripts. The method was applied to human normal mammary epithelial and breast cancer cells. Experimental validation of the presence of the sense and antisense transcripts by various techniques (e.g., northern blots, RT-PCR) supports the specificity of the method. When the antisense RNAs were specifically targeted, their corresponding mRNA levels significantly altered, a result consistent with a regulatory role for the identified antisense RNAs.