The monoclonal antibody Ki-S2 binds to a recently characterized proliferation-specific protein, p100. To assess its distribution pattern under physiologic and pathologic conditions, we performed immunohistochemical analyses on an exhaustive spectrum of normal tissues, 624 miscellaneous solid cancers, and 95 hematologic malignancies, and compared the results with Ki-67 immunostaining on consecutive sections. In addition, Ki-S2 expression was related to the DNA content by dual parameter flow cytometric analysis in parallel with Ki-67 labeling using a human cancer cell line. Immunoreactivity was enhanced at the G1/S transition and persisted through G2 and M phase. After adequate antigen retrieval, the antibody was found to yield identical results on fresh and formalin-fixed, paraffin-embedded material. The antibody specifically labeled actively proliferating cells, which constitute a subset of the population recognized by Ki-67. In normal human tissues, Ki-S2 immunolabeling hardly ever exceeded 40% of the Ki-67+ cell fraction. Immunoreactive scores of the two antibodies exhibited a linear correlation, but statistically significant differences in the ratio of Ki-S2-positive to Ki-67-positive cells were nevertheless observed between different tissue types. In contrast, the ratio of Ki-S2 and Ki-67 immunoreactive scores varied widely in neoplastic cells and tissues, occasionally attaining a ratio of almost 1:1. This suggests that loss of growth regulatory mechanisms in malignant cells might result in an extreme reduction of the G1 phase fraction and thus in a significantly shorter doubling time. Therefore, antibody Ki-S2 is likely to allow a more precise evaluation of the cell fraction that will complete a division cycle and a more confident appraisal of the malignancy potential of a neoplastic process.