Biochemical and molecular genetic studies were made on a case of placental aromatase (P-450AROM) deficiency. Of the enzymes participating in the electron transport system of placental microsomes, only aromatase activity was decreased specifically in the patient, being less than 0.3% of the normal activity. Northern and Western blotting analyses showed that the transcription of the aromatase gene and the translation of its mRNA proceeded normally in the placenta of this patient. However, aromatase cDNA isolated from a placental cDNA library of the patient was found to have an insert of 87 base pairs, encoding 29 amino acids in frame with no termination codon. The insert was located at the splicing point between exon 6 and intron 6 of the normal aromatase gene, and the extra DNA fragment was the first part of intron 6, except that its initial GT was altered to GC. These findings indicated that in this patient with aromatase deficiency, splicing between exon 6 and intron 6 did not occur at the normal position because of a point mutation in its consensus sequence and was forwarded to GT in the next cryptic consensus sequence 87 base pairs downstream according to the canonical GT/AG rule, resulting in translation of an abnormal protein molecule with 29 extra amino acids. During the transient expression in COS-7 cells, the aromatase cDNA of the patient was found to produce a protein with a trace of activity. This is the first report of a genetic defect for aromatase deficiency.