Sea urchin sperm before fertilization possess the longest nucleosome repeat length yet determined for any chromatin. By the time the fertilized egg gives rise to a blastula or gastrula embryo, the chromatin has a considerably shorter repeat length and, in addition, a sequence of different histone variants of H1, H2A, and H2B has appeared. We have investigated the relationship between these variations in histone composition and concomitant alterations in chromatin structure during the earliest stages of embryogenesis in two species of sea urchin. In contrast to the long repeat distance in sperm, chromatin loaded with cleavage stage histones has a much smaller repeat. Later stages containing predominantly alpha histones display an intermediate spacing. More detailed analysis of the events in the first cell cycle was carried out with polyspermically fertilized eggs. During the first 30 min after fertilization, in which sperm-specific H1 is completely replaced by cleavage-stage H1, the male pronuclear repeat remains unchanged. The decrease toward the repeat length of cleavage stages begins at about the time of DNA synthesis. Higher degrees of polyspermy extend the length of the cell cycle, including the duration of S phase and the length of time to reach the first chromosome condensation. At these higher degrees of polyspermy, the decrease in repeat length is also slowed. We conclude that the adjustment of the arrangement of nucleosomes in embryonic chromatin from that found in sperm can occur within the first cell cycle and that its timing is cell-cycle dependent. The adjustment is separable from a corresponding change in H1 composition.