In Drosophila, male reproductive fitness can be affected by any number of processes, ranging from development of gametes, transfer to and storage of mature sperm within the female sperm storage organs, and utilization of sperm for fertilization. We have previously identified the 89B cytogenetic map position of D. melanogaster as a hub for genes that effect male paternity success when disturbed. Here, we used RNA interference to test 11 genes that are highly expressed in the testes and located within the 89B region for their role in sperm competition and male fecundity when their expression is perturbed. Testes-specific knockdown (KD) of bor and CSN5 resulted in complete sterility, whereas KD of CG31287, Manf and Mst89B, showed a breakdown in sperm competitive success when second to mate (P2 < 0.5) and reduced fecundity in single matings. The low fecundity of Manf KD is explained by a significant reduction in the amount of mature sperm produced. KD of Mst89B and CG31287 does not affect sperm production, sperm transfer into the female bursa or storage within 30 min after mating. Instead, a significant reduction of sperm in female storage is observed 24 h after mating. Egg hatchability 24 h after mating is also drasti-cally reduced for females mated to Mst89B or CG31287 KD males, and this reduction parallels the decrease in fecundity. We show that normal germ-line expression of Mst89B and CG31287 is needed for effective sperm usage and egg fertilization.