Oviposition site preferences of Drosophila nigrospiracula and D. mettleri were determined for cactus versus soaked-soil substrates, light versus dark areas, and upper versus lower position in an attempt to explain the behavioral basis for the separation of their larval niches. The results show that D. nigrospiracula females discriminate oviposition sites mainly on the basis of substrate type, while D. mettleri females use substrate type and position as the primary basis of their site preference. These conclusions are supported by field data. Viability studies showed that D. nigrospiracula larvae cannot survive in the soaked-soil substrate while D. mettleri larvae survive equally well in either substrate. The evolution of soil-breeding is discussed, and the distinction is made between primary and secondary preferences in the case of D. mettleri with the soaked-soil/saguaro ecosystem.