The mode of inheritance of the 15 electrophoretically different α-naphthyl acetate-specific esterases found in five population samples of Drosophila montana collected near Gothic, Colorado, was delineated by mating a strain which exhibited no esterase activity to 237 wild-caught flies, and analyzing the progeny. This analysis showed that the α-esterases conform to a four-locus model with a high frequency of a null allele at each locus. Over 90% of the flies had an active esterase produced at only three or four of the possible eight loci; 75% of the chromosomes had two active alleles, presumably the optimum number. Extremely similar frequency distributions of the common allozymes were observed in all populations studied and over 3 years. From this analysis the frequencies of chromosomes of different allozyme types could be calculated. From these chromosome frequencies the probability of an active or a null allele at each locus was calculated. This, in turn, allowed determination of whether the presence of an active or null allele at one locus was independent of the activity at another locus. Analysis indicates extreme linkage disequilibrium between the lumped active and null alleles of loci 1 and 2, and between loci 3 and 4, the nature of the disequilibrium being the presence of an excess of chromosomes with an active allele at either locus 1 or 2 and an active allele at either locus 3 or 4. It is postulated that loci 1 and 2 specify one essential esterase (Ex) and loci 3 and 4 another (Ey). A crossover study showed that the four loci were located within a total distance of less than 1 centimorgan, and that their linear order was 1, 3, 2, 4. Linkage disequilibrium occurred between alleles of all pairs of loci except between alleles of the outer pairs (1-3 and 2-4), which were in linkage equilibrium. The polymorphism observed between active and null esterase alleles appears to be the result of two selective balancing forces: a chromosome with only two of the four loci active has a selective advantage; there is an advantage to a chromosome which produces both essential esterases, one produced by locus 1 or 2 and the other by locus 3 or 4. It is postulated that the four loci are evolutionarily related through a process of duplication of the original locus, then divergence in activity, followed by a tandem duplication of the previous duplication, to give the present genetic constitution 1Ex 3Ey 2Ex 4Ey.