The cDNAs for variant estrogen receptor (ER) mRNAs previously identified in human breast cancer biopsy samples have been cloned and characterized. Some of these cDNAs are unique to a tumor sample (e.g. clones 24 and 5), while others are present in multiple breast tumor samples (e.g. clone 4). The 5' ends of the variant cDNAs are essentially identical to sequences present in exons 1, 2, and 3 of the normal ER mRNA. However, at points which mark either the exon 2/intron or exon 3/intron boundaries, the variant cDNA sequences diverge and are unrelated to the normal ER mRNA. The unique sequences of clones 24 and 5 are unknown, and the unique sequence of clone 4 is related to the long interspersed repetitive LINE-1 sequences. The variant mRNAs contain open reading frames which could encode proteins containing known functional domains of the normal ER but missing others. In particular, the hormone binding domain of the normal ER is always missing. Furthermore, some of the variant transcripts may encode other unique proteins. In transient expression assays the proteins encoded by the variant ER mRNAs are unable to activate transcription of an estrogen-responsive reporter gene; neither are they able to modulate the ability of normal ER proteins to activate transcription.