We investigated dispersal patterns of Drosophila larvae searching for pupation sites over three substrates to determine the role of spatial heterogeneity and presence of other species on prepupation behavior. We used D. melanogaster, D. hydei, and D. pavani whose parents emerged from apples collected in one orchard. Each species showed different preferences for substrates on which to pupate, particularly in the presence of another Drosophila species. Larval locomotion rate and turning behavior in D. melanogaster, D. hydei, and D. pavani were modified depending this upon the type of substrate (agar and sand) on which the larvae crawled. These two behaviors are involved in dispersal and aggregation of pupae. Distance between pupae of the same species decreases when larvae of another species pupate on the same substrate. Aggregated distributions over the substrates lead to patches with few or no individuals. These could serve as pupation sites for other Drosophila species that, in nature,