Chromosomes in terminally differentiated mammalian spermatozoa are extensively condensed by protamines but a small proportion of histones remain. We examined the primary organization of somatic-type chromatin in lysolecithin-permeabilized human sperm nuclei and report that nucleosomes are closely packed with a periodicity of approximately 150 bp. Incubation of nuclei in the presence of exogenous Mg2+ and ATP induced chromatin reorganization leading to an increase in spacing of the nucleosomes to approximately 190 bp. This ATP-dependent chromatin rearrangement involved phosphorylation of both protamine and histone H2a. Increase in linker length between nucleosomes correlated with the phosphorylation of H2aX, the major H2a variant in human spermatozoa, predominantly at the C-terminal end. Chromatin reorganization was independent of detectable nuclear dispersion, which is an early chromosomal event in male pronuclear formation during fertilization.