Isofemale strains of Drosophila melanogaster and D. simulans were collected from wet and dry habitats in Tunisia. These strains were used to study the effect of habitat (collection site) and laboratory rearing temperature on larval pupation behaviour of these species. Results showed that D. melanogaster isofemale strains from the wet habitat pupated significantly higher in vials than those from the drier habitat. In contrast, the pupation behaviour of D. simulans isofemale strains was not affected by habitat type. Significant intraspecific variation in pupation behaviour was found for both species from both habitats. The distance pupated away from food in vials was positively correlated to the distance pupated from fruit in a field-like assay. Plasticity for pupation height was found at 16 and 32 °C for both species. Greater between-strain variation in pupation height was found for D. melanogaster at 32 °C and for D. simulans at 16 °C. The patterns of interspecific differences in larval pupation height paralleled those previously reported for adult behaviour patterns in these species.