The significance of amoebae for studies of nuclear architecture has considerably increased in the recent years. The availability of a protocol for isolation of nuclei in a quality sufficient for high-resolution light and electron microscopy is a prerequisite for such studies. Here we present a protocol for high enrichment of nuclei by sucrose density-gradient centrifugation. Moreover, we describe how to use these isolated nuclei as specimens for immunofluorescence and immune-electron microscopy of ultrathin sections.