DNA (from bacteriophage lambda or Xenopus) is assembled into nucleus-like structures when mixed with an extract from Xenopus eggs. Electron microscopy shows that these in vitro-reconstituted nuclei possess complete double membranes; some, but not all, nuclei have pore complexes. Extracts depleted of their endogenous ATP (by addition of ATPases) cannot assemble nuclear envelopes visible by phase-contrast microscopy. Once synthetic nuclei are assembled, however, they are stable when ATP is subsequently depleted, although their chromatin becomes condensed. About one-fourth of the nuclei assembled in vitro from lambda DNA accumulate nuclear proteins such as nucleoplasmin. ATP depletion blocks nucleoplasmin accumulation both in vitro, in pre-assembled synthetic nuclei, and in vivo, in the nucleus of microinjected oocytes. However, nucleoplasmin previously accumulated by reconstituted nuclei or by the germinal vesicle in microinjected oocytes is retained after ATP depletion.