How do we know whether different mutations that affect a characteristic occur at the same locus (are allelic) or occur at different loci? In fruit flies, for example, white is an X-linked recessive mutation that produces white eyes instead of the red eyes found in wild-type flies; apricot is an X-linked recessive mutation that produces light orange eyes. Do the white and apricot mutations occur at the same locus or at different loci? We can use the complementation test to answer this question.

To carry out a complementation test on recessive mutations, parents that are homozygous for different mutations are crossed, producing offspring that are heterozygous. If the mutations are allelic (occur at the same locus), then the heterozygous offspring have only mutant alleles (a b) and exhibit a mutant phenotype:

If, on the other hand, the mutations occur at different loci, each of the homozygous parents possesses wild-type genes at the other locus (aa b⁺b⁺ and a⁺a⁺ bb); so the heterozygous offspring inherit a mutant allele and a wild-type allele at each locus. In this case, the presence of a wild-type allele complements the mutation at each locus, and the heterozygous offspring have the wild-type phenotype:

Complementation has taken place if an individual possessing two recessive mutations has a wild-type phenotype, indicating that the mutations are nonallelic genes. There is a lack of complementation when two recessive mutations occur at the same locus, producing a mutant phenotype.

When the complementation test is applied to white and apricot mutations, all the heterozygous offspring have light-colored eyes, demonstrating that white eyes and apricot eyes are produced by mutations that occur at the same locus and are allelic.