Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little is known for Drosophila melanogaster. Here we characterize three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame.