We have have studied the relationship between nucleolar function and size and cell doubling time in cancer cells. Seven human cancer cell lines characterized by different proliferation rates were used. Nucleolar functional activity was evaluated by measuring RNA polymerase I activity and expression of RNA polymerase I upstream binding factor (UBF), DNA topoisomerase I, and fibrillarin, three proteins involved in synthesis and processing of rRNA. Transcriptional activity of RNA polymerase I was strictly related to cell doubling time (r = -0.97; P < 0.001). The quantitative distribution of UBF, DNA topoisomerase I, and fibrillarin was evaluated on Western blots using specific monoclonal antibodies by densitometric analysis of autoradiographic signals. It was found to be directly related to RNA polymerase I transcriptional activity (r = 0.89, P = 0.008 for UBF; r = 0.95, P = 0.001 for DNA topoisomerase I; and r = 0.91, P = 0.004 for fibrillarin) and inversely related to cell doubling time (r = -0.87, P = 0.011 for UBF; r = -0.97, P < 0.001 for DNA topoisomerase I; and r = -0.91, P = 0.005 for fibrillarin). The nucleolar areas were measured by automated image analysis on toluidine blue-stained cells. The values of the stained nucleolar structures per cell were directly related to RNA polymerase I transcriptional activity (r = 0.94, P = 0.001) and inversely related to cell doubling time (r = -0.98, P < 0.001). The same area values of the nucleolar structures stained by toluidine blue were also closely related to the amount of UBF (r = 0.92, P = 0.003), DNA topoisomerase I (r = 0.98, P < 0.001), and fibrillarin (r = 0.95, P = 0.001), and to the in situ quantitative distribution of AgNOR proteins (r = 0.98, P < 0.001). Our results demonstrated that in cancer cells rRNA transcriptional activity and nucleolar size are inversely related to cell doubling time. Quantitative distribution of nucleolar structures within the cell represents a cytohistological parameter of the rapidity of cell proliferation.