We previously identified 20 different alternatively spliced estrogen receptor alpha (ERalpha) mRNAs that have deletions in various combinations of exons in breast cancer cell lines using a novel 'Splice Targeted Approach' [J. Steroid Biochem. Mol. Biol. 72 (2000) 249]. In the current study, we compared the frequency of alternatively spliced ERalpha variant expression in 35 reduction mammoplasty and 38 breast cancer tissues with known ERalpha, ERbeta status using this highly specific 'Splice Targeted Approach'. A total of 16 different alternatively spliced variants were identified that have deletions in various combinations of exons in normal, as well as cancer tissues. However, not all 16 variants were present in every tissue. The frequency and type of variants in normal and cancer tissues was significantly different. Majority of normal tissues expressed only single exon deletion variants with the exception of those in combination with exon 2Delta and 7Delta. Tumor tissues, on the other hand, showed increased frequency of multiple exon deletion mRNAs (P<0.019). In addition, cancer tissues also showed an increased frequency of all variants compared with normal tissues (P<0.044). Among the 16 variants, the dominant negative variant, exon 3Delta, showed the most significant increase in cancer tissues (P=0.000032). Specifically, we detected four different mRNAs that have exon 2 deletion-exon 2Delta; 2 and 4Delta; 2 and 5Delta; and 2, 4-5Delta in various combinations in both normal and cancer tissues. A large number of normal tissues expressed two transcripts-exon 2Delta and 2, 4-5Delta. The multiple exon deletion 2, 4-5Delta were predominant in cancer tissues. Only the single exon 3 deletion variant, exon 3Delta, was detected in normal tissues. Cancer tissues showed the presence of a double exon deletion variant, exons 3 and 7Delta, in addition to exon 3Delta. A small fraction of normal tissues showed exons 2-3Delta mRNAs, whereas, cancer tissues showed increased frequency of exons 2-3Delta expression in addition to a triple exon deletion variant, exons 2-3, and 5Delta. The expression of exon 4Delta; or 4 and 7Delta or both was equivalent in normal and cancer tissues. Exon 5Delta transcripts were present at very low levels in both normal and tumor tissues. A small percentage of cancer tissues but not normal tissues showed exon 6Delta mRNA. The presence of single, double, triple and quadruple exon deletion mRNAs, exon 7Delta; 7 and 4Delta; 7, 3-4Delta; 7, 3-5Delta, respectively, were detected in normal as well as cancer tissues. Each normal and cancer tissue had a distinct profile of ERalpha wild type, ERbeta wild type and ERalpha splice variants. Heterogeneity in ER isoform profiles may result in variations in estrogen/anti-estrogen binding and activation/inactivation of estrogen-dependent genes, and, therefore, may have implications in the risk of developing breast cancer, survival with the disease and response to anti-estrogen, as well as other therapies.