Increasing evidence in the medical literature suggests that freezing a sample before fixation causes changes in the histologically observed mitotic activity. In a recent study we determined quantitative thresholds for mitotic counts in invasive ductal breast cancer in both nonfrozen and frozen formalin-fixed specimens. Survival- and recurrence-based analyses of this study material indicated grading thresholds of 17 and 32 mitoses/mm(2) for standardized mitotic index (SMI) and 13 and 35 mitoses/10 high-power fields for mitotic activity index (MAI). The purpose of the present study is to confirm and adjust the introduced thresholds in only nonfrozen formalin-fixed samples. The SMI and MAI in 202 cases of nonfrozen formalin-fixed samples were analyzed to determine optimal cutpoints for prognostication of invasive breast cancer on the basis of mitotic activity. The SMI thresholds were identical in both the present nonfrozen specimens and the previous combined specimens. The optimal MAI thresholds in the nonfrozen material changed to 11 and 37 mitoses/10 high-power field. The confirmation and adjustment of the mitotic thresholds improved the prognostic significance of the method in the nonfrozen material, which will contribute to the clinical applicability of a morphometric grading system.