Abstract Unlike oocytes (and all somatic cells), which package their DNA with histones, the DNA of mature sperm is bound by protamines, a highly basic and rapidly evolving protein that is essential for compaction of the paternal genome. This differential packaging of egg and sperm DNA traces back >500 million years, yet our understanding of the principles of genome organization remains poorly understood. To examine this fundamental yet poorly understood process we have developed novel genetic and biophysical tools to identify proteins implicated in the histone-to-protamine exchange and to dissect the molecular mechanisms of chromatin reorganization during spermiogenesis. In preliminary studies we developed epitope-tagged protamine 1 and 2 mice and relevant reagents such as specific antibodies. Immunoprecipitation followed by mass spectrometry has identified multiple potential chromatin remodelers/chaperones, which we will functionally characterize in this proposed study, both in vivo and in vitro. Since protamine packaging results in a super-condensed chromatin state, which does not easily lend itself to mechanistic investigations in vivo, we have developed novel methods to purify protamines from sperm to use in both in vitro DNA curtain assays and in vitro reconstitution assays of the histone-to-protamine exchange process. The overall goal of this proposal is to apply our diverse suite of genetic, molecular, and biophysical tools to gain mechanistic insights into normal sperm chromatin packaging, which is essential for genome fidelity and reproductive fitness of the species.