p330d, also known as CENP-F, is a newly characterized cell cycle specific nuclear autoantigen which is associated both with the centromeres and the nuclear matrix. It is expressed in low amounts in G0/G1-cells and accumulates in the nuclear matrix during S-phase with a maximum expression in G2/M-cells. In the present study we have investigated if p330d/CENP-F could be used as a marker for proliferation in different human malignancies. A flow cytometric method was developed by which p330d/CENP-F expression and DNA-content could be assessed on hematopoietic and solid tumors. Twenty-four different human hematopoietic malignancies, 12 breast cancers, and several cell lines were analyzed and the number of p330d/CENP-F positive cells and the S-phase fraction were determined. The percentage of p330d/CENP-F positive cells correlated with the fraction of S-phase cells in all human malignancies tested. Various cell lines revealed a similar cell cycle specific distribution. The association of p330d/CENP-F with the nuclear matrix facilitated the flow cytometric analysis of this protein due to its resistance to different preparation and fixation procedures. In summary, p330d/CENP-F seems to be a potentially valuable proliferation marker which can be applied to different tumors.