Increasingly, biochemical analyses of processes that occur within eukaryotic nuclei such as transcription and replication require the construction of specific chromatin substrates. Nucleosome complexes reconstituted in vitro have been key elements in a variety of recent studies of polymerase progression and trans-acting factor binding activities. Reconstituted complexes can be easily constructed from purified components in quantities suitable for biochemical and biophysical studies. In addition, reconstituted mononucleosome complexes exhibit native biochemical and biophysical properties but necessarily contain much less heterogeneity with regard to both protein and DNA components than bulk complexes isolated from natural sources. This review details the protocols for reconstitution of model mononucleosome complexes that contain unique DNA sequences and specifically tailored core histone proteins and describes common pitfalls associated with these procedures.