Hybridization experiments and cuticular hydrocarbon analyses were performed to determine the extent of the reproductive isolation present to clarify the taxonomic status of Danaus erippus (Cramer, 1775), which has often been classified as a subspecies of Danaus plexippus (Linnaeus, 1758). The results of crosses between D. erippus and Danaus plexippus nigrippus (Haensch, 1909) showed pre- and postzygotic isolation. Pupal inviability, and Haldane rule effects (male hybrids only, the homogametic sex in butterflies) were observed after the crosses. These results reinforce the hypothesis that D. plexippus and D. erippus are separate, reproductively isolated species. However, we have also investigated the cuticular hydrocarbons in D. erippus and D. p. nigrippus with temperature-programmed gas chromatography. The chromatographic profiles of peaks were similar in both taxa, with retention indices in the range 2100-3797. The profiles were characterized by the presence of 15 peaks whose retention indices were in the range 2501-3772. Although no major qualitative differences between the two species were observed, we did find some quantitative differences. A principal component analysis showed a clear separation between members of D. erippus and D. p. nigrippus, suggesting that semiochemical differences might have become differentiated after isolation, perhaps in response to different physiological pressures. © 2007 The Linnean Society of London.