CENP-F is a newly characterized cell cycle-associated nuclear antigen that is expressed in low amounts in G0/G1 cells and that accumulates in the nuclear matrix during S phase with a maximal expression in G2/M cells. CENP-F can be analyzed by flow cytometry and used as a proliferation marker. In the present study, therefore, we characterized the expression of CENP-F in non-Hodgkin's lymphoma by immunohistochemical techniques to detect potential dysregulation of the protein or to establish CENP-F as a reliable proliferation marker. A polyclonal rabbit antibody reacting with CENP-F was prepared and used for immunohistochemical analyses after antigen retrieval. The rabbit antibody produced immunofluorescence patterns, flow cytometric profiles, and Western blot reactivity identical to those of the human autoantibody used in earlier studies. The percentage of CENP-F-positive and Ki-67-positive cells, as well as the labeling index, S-phase time, and potential doubling time, derived from in vivo iododeoxyuridine incorporation, were evaluated in 41 non-Hodgkin's lymphomas. Aggressive lymphomas showed higher CENP-F values than did indolent cases (10.1 vs. 3.4%). The percentage of CENP-F-positive cells correlated significantly to the S-phase fraction (r(s) = 0.68), the Ki-67 index (r(s) = 0.56) and the labeling index of iododeoxyuridine (r(s) = 0.47), as well as to S-phase time and potential doubling time (r(s) = 0.34 and -0.40). A lower fraction of CENP-F-positive cells was found, compared with the Ki-67 index (4.9 vs. 9.4%), supporting previous observations that CENP-F was expressed in a fraction of actively growing cells. These correlative data indicate that CENP-F expression defines a specific subpopulation of growing cells and that no clear evidence for dysregulation was found. Accordingly, CENP-F seems to be a useful proliferation marker for formalin-fixed and paraffin-embedded material.