Semen collection and preservation is the first step toward the development of an artificial insemination program in endangered Pteropus spp. Semen was collected by manual stimulation from a single "human-habituated" P. alecto. Manual stimulation resulted in the successful collection of motile spermatozoa on 17 of 34 attempts. The semen had a pH of 8.2 (n=2). With the exception of volume, seminal characteristics (concentration, motility, acrosome and plasma membrane status) were similar to those collected previously by electro-ejaculation. Zoo Biol 27:159-164, 2008. (c) 2008 Wiley-Liss, Inc.