Periodic arrays of nucleosomes were assembled on heterogeneous DNA using core histones, the histone chaperone NAP-1, and ATP-dependent chromatin assembly and remodeling factor (ACF). The mechanical properties of these complexes were interrogated by stretching them with optical tweezers. Abrupt events releasing approximately 55-95 base-pairs of DNA, attributable to the non-equilibrium unraveling of individual nucleosomes, were frequently observed. This finding is comparable with a previous observation of 72-80 bp unraveling events for nucleosomes assembled by salt dialysis on a repeating sea urchin 5 S RNA positioning element, but the unraveling force varied over a wider range ( approximately 5-65 pN, with the majority of events at lower force). Because ACF assembles nucleosomes uniformly on heterogeneous DNA sequences, as in native chromatin, we attribute this variation to a dependence of the unraveling force on the DNA sequence within individual nucleosomes. The mean force increased from 24 pN to 31 pN as NaCl was decreased from 100 mM to 5 mM. Spontaneous DNA re-wrapping events were occasionally observed in real time during force relaxation. The observed wide variations in the dynamic force needed to unravel individual nucleosomes and the occurrences of sudden DNA re-wrapping events may have an important regulatory influence on DNA-directed nuclear processes, such as the binding of transcription factors and the movement of polymerase complexes on chromatin.