GAL4-VP16-mediated nucleosome reconfiguration and transcriptional activation were observed with preassembled chromatin templates that contained regular and physiological nucleosome spacing. Both processes were dependent on adenosine triphosphate (ATP), although binding of GAL4-VP16 to the chromatin was ATP-independent. Factor-mediated nucleosome reconfiguration was not, however, sufficient for transcriptional activation. These experiments recreate in vitro the active participation of nucleosomal cores in the regulation of transcription that occurs in vivo, and they suggest a multistep pathway for transcriptional activation in which factor- and ATP-dependent nucleosome reconfiguration is followed by facilitation by the DNA-bound activator of transcription from the repressed chromatin template.