A subgroup of 10–15% of Caucasians are termed phenotypical ‘intermediate metabolizers’ of drug substrates of CYP2D6 because they have severely impaired yet residual in-vivo function of this cytochrome P450. Genotyping based on the currently knownCYP2D6alleles does not predict this phenotype satisfactorily. A systematic sequencing strategy through 1.6 kb of theCYP2D65′-flanking sequence revealed six mutations of which three were exclusively associated with the functionalCYP2D6*2allele (−1496 C to G;−652 C to T; and−590 G to A), two were associated with the nonfunctional*4and with the functional*10-alleles (−1338 C to T and−912 G to A) and one (−1147 A to G) was seen in all*2,*4and*10-alleles investigated. The−1496 C to G mutation was found to be polymorphic withinCYP2D6*2alleles. In a family study, the wild-typeCYP2D6 *2[−1496 C]and the novel variant[−1496 G]allele co-segregated with lower and higher CYP2D6 in-vivo function, respectively, as shown by phenotyping using sparteine as probe drug. In a representative population sample selected for genotypes comprising oneCYP2D6*2and one non-functional allele, the median urinary metabolic ratio (MRs) for sparteine oxidation was 4.4-fold reduced in individuals with the variant allele (*2[−1496 G], MRs= 0.53,n= 27) compared with individuals lacking the mutation (*2[−1496 C], MRs= 2.33,n= 12;P<0.0001). The mutation−1496 C to G has an estimated frequency of approximately 20% in the general population and allows establishment of a genotype for the identification of over 60% of intermediate metabolizers in Caucasian populations.