NAP1 (nucleosome assembly protein 1) is a histone chaperone that has been described to bind predominantly to the histone H2A.H2B dimer in the cell during shuttling of histones into the nucleus, nucleosome assembly/remodeling, and transcription. Here it was examined how NAP1 interacts with chromatin fibers isolated from HeLa cells. NAP1 induced a reversible change toward an extended fiber conformation as demonstrated by sedimentation velocity ultracentrifugation experiments. This transition was due to the removal of the linker histone H1. The H2A.H2B dimer remained stably bound to the native fiber fragments and to fibers devoid of linker histone H1. This was in contrast to mononucleosome substrates, which displayed a NAP1-induced removal of a single H2A.H2B dimer from the core particle. The effect of NAP1 on the chromatin fiber structure was examined by scanning/atomic force microscopy. A quantitative image analysis of approximately 36,000 nucleosomes revealed an increase of the average internucleosomal distance from 22.3 +/- 0.4 to 27.6 +/- 0.6 nm, whereas the overall fiber structure was preserved. This change reflects the disintegration of the chromatosome due to binding of H1 to NAP1 as chromatin fibers stripped from H1 showed an average nucleosome distance of 27.4 +/- 0.8 nm. The findings suggest a possible role of NAP1 in chromatin remodeling processes involved in transcription and replication by modulating the local linker histone content.