The mouse knockout of the estrogen receptor alpha (ERalpha) gene, known as alphaERKO, has been extensively used for several years to study the role and function of ERalpha. Residual estradiol binding capacity in uterine tissue of 5-10% raised doubts if this knockout is a genuine null mutation of ERalpha. Although alternatively spliced ERalpha mRNA variants in the alphaERKO mouse were reported previously, the corresponding protein isoforms have not been detected to date. Here we show that a variant ERalpha protein, 61 kDa in size, is expressed in the uterine tissue of alphaERKO mice as a result of an alternative splicing. The transactivation capability of this protein is cell dependent and can be as high as 75% of the wild type ERalpha.