Immunogold staining combined with heavy metal-based DNA immunostaining of ultrathin sections of cryofixed, freeze-substituted, and resin-embedded cells has revealed that in the hyperthermophilic archaeon, Sulfolobus acidocaldarius, the histone-like proteins HSNP-A and HSNP-C' are colocalized with DNA in the ribosome-free nucleoid, whereas DBNP-B is located exclusively in the ribosome-containing cytoplasm. Following chemical fixation and solvent dehydration the chromatin was resistant to aggregation, indicative of a high protein content. Immunogold labeling of chromatin released from isolated nucleoids of S. acidocaldarius demonstrated that the helix-stabilizing protein HSNP-C' was randomly distributed on both filamentous DNA and on more globular material present within distinct nucleoid subdomains. DNA-dependent RNA polymerase molecules were located by immunogold staining predominantly in the cytoplasmic region immediately surrounding the nucleoid in S. acidocaldarius, presumably at the locations of active transcription. Affinity-purified antibodies raised against HMf, a histone-like protein isolated from the hyperthermophilic and only distantly related methanogenic archaeon Methanothermus fervidus, were also found to be localized almost exclusively to the DNA-containing, ribosome-free nucleoid region. (C) 1994 Academic Press, Inc.