We studied the deposition of pupae of the winged bat fly Trichobius sp. (caecus group; Diptera), an ectoparasite of Natalus stramineus (Chiroptera, Natalidae), in a natural cave in Tamaulipas, Mexico. For the first time, we show a strong spatial segregation of populations of a streblid bat fly at different stages of development. Using molecular techniques we were able to match developmental stages to adults. Only 5 pupae were present in the main bat roosts. The overwhelming majority occurred exclusively in the bat flyway passages at a considerable distance from roosting bats. Pupal density corresponded positively with the average flight height of bats in the cave passage. Taken together, observations suggest that these ectoparasites must actively seek out their hosts by moving onto passing or roosting bats. The scarceness of pupae in the main roost may be dictated by environmental constraints for their development. The estimated population of viable pupae far exceeds the population of imagoes on the bats, and predation on adults by spiders is common.