We have discovered two types of 5' intronic gene mutation that impair androgen receptor (AR) mRNA expression severely, and cause complete androgen insensitivity. Labium majus skin fibroblasts (LMSF) hemizygous for each mutation had negligible specific androgen binding, and did not react to an antibody against an N-terminal peptide of the AR. Both mutations were detected by direct sequencing of exons PCR-amplified with flanking primers. One mutation is an adenine to thymine transversion at position +3 of the intron 6 splice-donor site. Using LMSF mRNA, RT-PCR of a portion of the AR androgen-binding domain yielded a small amount of a 302-bp mutant fragment instead of a 433-bp wild-type product. Sequencing established that exon 5 was followed, out of frame, by exon 7: exon 6 was skipped. The other mutation is a thymine insertion at the +3 position of the intron 1 donor-splice site. RT-PCR and sequencing revealed a small amount of normal-size mRNA with normal exon 1-exon 2 splicing. Quantitative RT-PCR on mutant LMSF showed AR mRNA levels were well below 10% of normal; hence, most of the aberrant AR mRNA resulting from each mutation is probably unstable. The misbehavior caused by these two mutations indicates that in the AR the splice-donor site +3 adenine is critical; indeed, 57% of eukaryotic introns have adenine in the +3 position, while only 2% have thymine.