The rt locus in Ephestia (Anagasta 1 ) kuhniella is characterized by two alleles which control pigmentation of the testis sheath cells (Rt for brown-blue testis; rt for red testis). It is a reasonable assumption that both alleles occur in wild populations of this moth in North America and Europe although no specific allele-sampling study has been made in the natural populations to substantiate it. Caspari (1933, 1943) identified this allelic polymorphism initially in German strains, then again in American materials provided by Prof. P. W. Whiting. More recently (1950), he found that of two strains from Prof. A. Kiihn's laboratory, the first proved homozygous for the Rt allele, and the second homozygous for the rt allele. These observations, in the aggregate, imply that both alleles are to be found in samples from natural populations of this moth species.Phenotypic expression of the two alleles in pigment formation is restricted to the testis only; no other ommochrome-containing organ is affected (Caspari, 1943; Wolfram, 1949), and the genotype of the female for this locus can be inferred only by progeny testing. The biological basis for natural selection resulting in a balanced 1 Assignment of Ephestia kiihniella to the genus Anagasta was made by Heinrich (1956) on the basis of differences in morphology of this species when compared to the other species more typical of the original genus. However, a recent taxonomic study of R. V. Roesler (1973) reinstated the generic name Ephestia and assigned Anagasta as the subgenus. The established traditional name of this moth, Ephestia kilhniella, will be used throughout these reported genetic studies. polymorphism of the three possible genotypes must be sought in areas other than that of coloration because the differences in pigmentation are not visible without dissection of the male abdomen.Backcross and F2 cultures which were segregating for Rt and rt were analyzed by Caspari (1950) for differences in viability, speed of development, and mating behavior. The red testis male (rtlrt) was found to be significantly more viable than the blue-brown testis males (Rt/rt and RtlRt) of the F 2 cultures. In all other features the heterozygous male (Rtlrt) was equal, or slightly superior, to either homozygote. Analysis of mating pairs showed that significantly more heterozygous males were found in copula than would have been expected on the basis of their proportion in the population of males. These observations suggest a net positive selective value of the rt allele.This study was undertaken in order to relate changes in the rt allele frequency of laboratory populations with modifications of male reproductive behavior resulting from single allele substitution. It demonstrates 1) that there is a sustained polymorphism at the rt locus in population cages with an increase in the relative frequency of the rt allele, and 2) that the reproductive behavior of the rtlrt male Ephestia is a complex function of the relative genotypic composition of the population, of the adult male populat...