We have identified cuticular pheromones that sustain the integrity of the six Drosophila paulistorum semispecies. Hexane extracts of male and female cuticles were separated on a silica gel column and analyzed using gas chromatography/mass spectrometry. Both sexes of each of the six semispecies have the same fifteen major cuticular components, all hydrocarbons ranging from C31 to C37. However, all males have four additional ester compounds. Bioassay observations showed that this four-component ester complex imparts a strong anti-aphrodisiac effect on intra-semispecific mating behavior, thus confirming its pheromonal role. The three major ester components are methyl (Z)-9-tetradecenoate (C15H28O2), 11-docosenyl acetate (C24H46O2), and 19-triacontenyl acetate (C32H62O2). The fourth ester is a di-unsaturated acetate with molecular formula C32H60O2, but the positions of unsaturation have not been determined. Bioassays indicate that the male-specific complex of the transitional semispecies, the relict ancestor, imparts anti-aphrodisiac effects on the other semispecies as well, but effectiveness decreases with phylogenetic distances. Across the six semispecies, the male-specific compounds are the same, but vary quantitatively. Apparently, the quantitative differences among these incipient species act efficiently to preclude hybridization in nature. Because Drosophila paulistorum is a cluster of incipient species, this opportunity to observe pheromonal influences on speciation is unique.