During spermatogenesis, chromosomes are partitioned and specifically packaged to ensure transmission of the intact paternal genome to the next generation. This packaging not only protects the sperm DNA from damage, it also aids in transcriptional silencing and efficient sperm motility. Important paternal factors required for development, like SPE-11, are also tightly associated with sperm chromatin. Proteins that assemble or associate with sperm chromatin are vital for fertility, as specific cases of human male infertility result directly from defective sperm chromatin packaging. Sperm-specific chromatin packaging is a conserved event from worms to humans; however, little is known about the basic cellular components necessary to achieve it. We are conducting a large-scale identification and analysis of sperm chromatin-associated proteins that are important for sperm chromatin packaging and fertility in C. elegans. A subtractive proteomic approach comparing sperm and oocyte chromatin has initially identified over 200 sperm-specific chromatin-associated proteins. Interestingly, 21% (44 out of 202) of the proteins identified show homology to mouse or human proteins that are linked to roles in spermatogenesis, gametogenesis, or meiosis. Initial candidates for further characterization were selected through homology with proteins linked to either fertility in other organisms or chromatin organization. Interesting candidates have emerged from this analysis including C. elegans protamine candidates, specific histone variants, and chromatin-associated phosphatases and kinases. Temporal and spatial requirements for initial candidate proteins are being assessed by immunofluorescence to help define how proteins participate in sperm-specific functions. As an example, E03A3.4 is a histone H3 variant that localizes specifically between homologs in metaphase I and sisters in metaphase II of meiosis and around mature sperm DNA, a pattern distinct from the centromeric localization of another histone H3 variant, CENP-A. E03A3.4 also colocalizes with the Aurora-like kinase, AIR-2, that is involved in chromosome segregation, condensation, cohesion, and cytokinesis. This finding represents the first description of a histone H3 variant localizing specifically between homologs and sisters during sperm meiosis. Functional roles in sperm chromatin assembly, fertilization, and embryogenesis of E03A3.4 and other candidate proteins are currently being assessed through RNAi or mutant analysis.