The isolation and characterization of several new human estrogen receptor-a (hERa) mRNAs are de- scribed. Together with those previously identified, they give rise to a total of six hERa mRNA isoforms (A-F hERa mRNAs). Produced from a single hERa gene by multiple promoter usage, all these tran- scripts encode a common protein but differ in their 5*-untranslated region as a consequence of alter- native splicing of five upstream exons (1B-1F). RT- PCR and S1 nuclease mapping analysis of these different hERa mRNA isoforms revealed a differ- ential pattern of expression of the hERa gene in human tissues and cell types. The A hERa mRNA is the main isoform detected in mammary glands or in the tumor cell lines derived from this tissue. In endometrium, the predominant forms are the A and C hERa mRNA isoforms, whereas the C and F hERa mRNA isoforms are the major forms de- tected in ovary. Finally, high levels of the E hERa mRNA isoform are restricted to the liver with an increased expression in females. Taken together, our results demonstrate that the hERa gene is a complex genomic unit exhibiting alternative splic- ing and promoter usage in a tissue-specific man- ner. (Molecular Endocrinology 12: 1939-1954, 1998)