Transitions in the histone complement of nuclei during sea urchin spermatogenesis were investigated by two-dimensional gel electrophoresis. Nuclei were isolated from male gonads of individuals differing in degree of maturity. Unlike protamines, the sea urchin sperm-specific histone variants Sp H1 and Sp H2B appear early in spermatogenesis, well before spermatid differentiation, as the predominant representatives of their classes. Both proteins are phosphorylated from their first appearance until the last steps of spermiogenesis, when the highly condensed late spermatid nuclei become spermatozoan nuclei. Phosphorylation of serine occurs mostly (Sp H1) or entirely (Sp H2B) on the N-terminal portions of these molecules. We conclude that phosphorylated sperm-specific histone variants in the sea urchin function in spermatocytes during meiosis and are the major histones present during replication and transcription in some spermatogonia as well. We propose that the dephosphorylation of Sp H1 and Sp H2B in late spermatids is not primarily responsible for spermatid chromatin condensation but instead may act to stabilize the chromatin of the spermatozoon or aid in the final shaping of the sperm nucleus.