Recombinant yeast nucleosome assembly protein (yNAP-1) facilitates the formation of uniformly spaced nucleosomes from high molecular weight DNA and core histone octamers. No additional factors or metabolites are required. The repeat length of the chromatin produced is about 146 base pairs. To obtain the most distinct nucleosomal ladders, the core histones must preexist as an octamer complex. yNAP-1 forms complexes with core histones as judged by native gel electrophoresis, chemical cross-linking, limited histone proteolysis, and affinity blotting. A discrete complex was observed with a probable ratio of yNAP-1 to histone octamer of 4:1. Chromatin produced by salt dialysis does not contain uniformly spaced nucleosomes, but subsequent incubation with yNAP-1 creates uniform spacing. Trypsin-treated core octamers that lack amino termini, although capable of forming core particles with core-length DNA by salt dialysis, are not assembled by yNAP-1 into uniformly spaced nucleosomes on high molecular weight DNA. Proteolytic removal of the amino termini of the core histones precludes complex formation between a histone octamer and yNAP-1. Affinity blotting also demonstrates that yNAP-1 binds linker histones and high mobility group (HMG)-1/HMG-2 but not HMG-14. Competition experiments with poly-L-arginine, poly-L-lysine, and protamine reveal that yNAP-1 binds to core and linker histones more tightly despite the much higher positive charge densities of the former molecules. Naturally occurring acetylated histone H4 species show no evidence for differential yNAP-1 binding. yNAP-1 is not bound tightly to the resulting chromatin after deposition and thus could act catalytically.