Androgen insensitivity in the testicular feminized (Tfm) mouse is caused by frame-shift mutation in the androgen receptor (AR) mRNA, which results in a stop codon in the amino terminus. Despite this mutation, a smaller sized protein corresponding to the DNA- and steroid-binding domain of the AR can be synthesized from the cloned Tfm AR cDNA by in vitro translation. The Tfm AR construct was demonstrated to express a protein capable of binding androgen with an affinity similar to the cloned wild-type AR. Although the Tfm AR product failed to transactivate mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter when low concentrations (100 ng) of Tfm AR vector were cotransfected, higher concentrations (5000 ng) resulted in a residual amount of transactivation, suggesting lower level transactivating capabilities. By cotransfecting the Tfm AR expression vector with the wild-type receptor, it was demonstrated that the product of the Tfm AR gene is capable of inhibiting the transactivation activity of the wild-type receptor. These data suggest that although the Tfm AR mRNA fails to produce a full-length AR because of the frame-shift mutation, a smaller protein capable of binding both steroid and DNA can be produced by translation from an internal initiation codon. This product could account for the low levels of androgen-binding activity detected previously in the Tfm mouse.