From a human fetal brain cDNA library, we isolated a novel gene sharing significant homology with the genes of nucleosome assembly proteins (NAPs). This cDNA clone, designated NAP1L3 (nucleosome assembly protein 1-like 3), contained an open reading frame of 1518 nucleotides encoding 506 amino acids. Its predicted amino acid sequence showed 46% identity and 65% similarity with NAP1L. In its C-terminal half NAP1L3 contained several characteristic motifs strictly conserved with NAP1L and yeast NAP1, but the N-terminal half showed little conservation. Northern-blot analysis revealed strong expression of a 3.0-kb transcript in human adult brain and weak expression in heart. NAP1L3 was closely linked to a marker (DXS990) mapped to chromosome bands Xq21.3-->q22, where genes responsible for several X-linked mental retardation syndromes have been localized.