Central to the study of speciation is the identification of the isolating mechanisms that reduce gene flow. Of special interest are those isolating mechanisms that are the earliest to arise. Sperm fate and function play a major role in two distinct types of reproductive isolation in the genus Drosophila. First, postcopulatory-prezygotic (PCPZ) incompatibilities between the male ejaculate and a heterospecific female reproductive tract can result in a reduction in sperm fertilisation success. Although PCPZ incompatibilities have, until recently, received little attention, overwhelming diversity in reproductive morphology and biochemistry indicates that they may play an important role in speciation in this genus. The second type of isolating mechanism that affects sperm occurs in the testes of hybrid males produced by heterospecific matings. These individuals often suffer from dysfunction in spermatogenesis, resulting in the production of aberrant sperm. Hybrid male sterility and hybrid inviability are examples of postzygotic (PZ) reproductive isolation. The observation that hybrid sterility is pervasive among males of all taxonomic groups is known as Haldane's rule. Here we discuss both the evolutionary origins, and functional causes of both PCPZ incompatibilities, which affects sperm fate and function in females, and one type of PZ incompatibility, hybrid male sterility, or sperm dysfunction in hybrid males. Although these two mechanisms of isolation are quite distinct, they are similarly caused by breakdowns in epistatic interactions which occur in the encounter between two divergent genomes. Molecular, cytological, and empirical data are discussed, as is relevant evolutionary theory.