The behavioural mechanisms by which the larvae of the sibling species D. pavani and D. gaucha, and their reciprocal hybrids select pupation sites are described in terms of larval substrate preferences. If the larval behaviour of the parental species and their reciprocal hybrids during pupation leads them to pupate in different substrates, such preferences could be used to identify environmental factors and genetic mechanisms involved in the evolution of that behaviour. Confronted with dry substrates, interspecific hybrid larvae pupated in the rearing cup, while the parental species pupated both outside and inside the cup. Like the parental species, the hybrids pupated outside, when humid substrates surrounded the rearing cup. Larvae from D. pavani and D. gaucha showed a broader norm of reaction than the hybrids. D. pavani larvae occupied dry and humid substrates to pupate, while D. gaucha larvae tended to prefer only humid substrates. Larvae of D. pavani burrowed into dry sand pupated preferently in the deep layers of the substrate. Few D. gaucha larvae burrowed; they tended to pupate in the upper layers of dry sand. D. pavani larvae pupated on the moist sand, while most of D. gaucha larvae burrowed and formed puparia in the upper layers of the substrate. The hybrid larvae showed no burrowing behaviour in either dry or moist sand.