It has been shown recently that the overall metabolism of amobarbital in man is essentially under genetic control. The drug normally undergoes two hydroxylation reactions, leading to 3'-hydroxyamobarbital (C-OH) and N-hydroxyamobarbital (N-OH). This paper describes a sibship in which two mothers who are identical twins show a gross deficiency on N-OH elimination in urine. The whole set of sibship data suggests that this deficiency represents a recessive trait controlled by a single pair of allelic autosomal genes which regulate N-OH formation. Several methodical approaches to assess an individual's capacity for N-OH formation are illustrated. There was no evidence of compensatory or concordant regulation of the two hydroxylation reactions. The case of this family illustrates that the functional lack of a biotransformation reaction is almost certain to be overlooked if one measures only the disappearance of a multimetabolized drug and not the appearance of metabolites.