The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed in Chinese hamster ovary cells using specific antibodies to this protein and immunocytochemical techniques. By immunofluorescence and immunocytochemistry at ultrastructural level in situ, the 100 K protein was detected abundantly in the nucleolus of interphase cells. In exponentially growing cells, the dense fibrillar component was shown to contain more 100 K protein than the granular RNP component but both the nucleolar components were positively immunostained. Fibrillar centers consistently showed weaker or no staining. The 100 K association with the preribosomal RNP components of the interphase nucleolus supports evidence for a role of this protein in pre-rRNA transcription and preribosomal processing.