A critical feature of chromatin with regard to structure and function is the regular spacing of nucleosomes. In vivo, spacing of nucleosomes occurs in at least two steps, but the mechanism is not understood. In this report, we have mimicked the two-step process in vitro. A novel spacing activity has been partially purified from Xenopus laevis ovaries. When this activity is added, either at the beginning or at the end of a nucleosomal assembly reaction, it can convert a DNA template consisting of irregularly spaced nucleosomes into a chromatin structure made up of regularly spaced nucleosomes with a repeat length of about 165 base pairs. The reaction requires ATP. Histone H1 is able to increase the nucleosomal repeat from 165 to 190 base pairs. This two-step increase in nucleosomal repeat length suggests that both the spacing activity and histone H1 contribute to generating repeat lengths of greater than 165 base pairs and that their contributions may be additive. Alternatively, the critical step in the spacing reaction may not be the formation of the 165-base pair repeat but may be the sliding of nucleosomes or the reorganization of the octamer structure induced by the spacing activity.