Chromatin fibers have been hydrolyzed by trypsin and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and scanning force microscopy (SFM). At early points of hydrolysis, when mostly only the tails of the linker histones have been cleaved, nucleosomes appear to pile upon each other within a fiber. Later, once significant hydrolysis of the N-terminal tail of histone H3 has occurred, fibers exhibit an open, three-dimensional arrangement of nucleosomes. Linker DNA between adjacent nucleosomes is observed for the first time. Adjacent nucleosomes appear to have a 'zig-zag' arrangement. Finally, when all the tails of the linker histones and the N-terminal tails of H3 have been cleaved, then the fibers exhibit (i) a flat two- dimensional arrangement of nucleosomes, (ii) linker DNA between nearly all nucleosomes, and (iii) a zig-zag arrangement among some nucleosomes. We suggest that (i) the linker histone globular domains help fix the angle of the DNA entering and exiting the nucleosome, (ii) the angle, however, is not sufficient to maintain the three-dimensionality of the fiber, and (iii) the N-terminal tails of histone H3 ares necessary for the three-dimensional conformation of the fiber at low ionic strength.