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We describe the cloning and analysis of Drosophila nucleosome assembly protein 1 (dNAP-1), a core histone-binding protein that functions with other chromatin assembly activities in a Drosophila chromatin assembly factor 1-containing fraction (dCAF-1 fraction) in the ATP-facilitated assembly of regularly spaced nucleosomal arrays from purified core histones and DNA. Purified, recombinant dNAP-1 acts cooperatively with a factor(s) in the dCAF-1 fraction in the efficient and DNA replication-independent assembly of chromatin. In the presence of histone H1, the repeat length of the chromatin is similar to that of native chromatin from Drosophila embryos. By coimmunoprecipitation analysis, dNAP-1 was found to be associated with histones H2A and H2B in a crude whole-embryo extract, which suggests that dNAP-1 is bound to the histones in vivo. Studies of the localization of dNAP-1 in the Drosophila embryo revealed that the factor is present in the nucleus during S phase and is predominantly cytoplasmic during G2 phase. These data suggest that NAP-1 acts as a core histone shuttle which delivers the histones from the cytoplasm to the chromatin assembly machinery in the nucleus. Thus, NAP-1 appears to be one component of a multifactor chromatin assembly machinery that mediates the ATP-facilitated assembly of regularly spaced nucleosomal arrays.