Recent studies suggest that proliferative activity (S-phase fraction [SPF]) may have greater prognostic significance than total nuclear DNA content; however, relatively few studies have examined SPF from paraffin-embedded tissue because of significant contamination of histograms with debris. In this study, cell cycle analysis was performed on 124 matched tissue specimens. Fresh tissue was divided into two equal portions; one portion was frozen, whereas the other portion was processed and embedded in paraffin. S-phase could be determined for both frozen and paraffin-embedded tissue in 81 cases. Correlation between SPF from frozen and paraffin-embedded tissue was demonstrated (r = 0.80) when debris was subtracted from histograms with the use of two new subtraction algorithms referred to as multicut and singlecut. Unlike other debris-subtraction algorithms, the quantity and distribution of debris calculated by these algorithms are dependent on the magnitude and position of histogram peaks. A lesser degree of correlation was demonstrated with the use of a standard exponential debris subtraction algorithm (r = 0.67). Correlation of SPF for aneuploid cases was greater when SPF was calculated as a percentage of the aneuploid cell population rather than as a percentage of the entire cell population. This was attributed to the observation that the proportion of aneuploid cells from paraffin-embedded tissue was less than that from frozen tissue. The results of this study indicate that SPF can be calculated from paraffin-embedded tissue with values comparable to those obtained from frozen tissue. The ability to calculate SPF reliably from paraffin-embedded tissue should allow additional evaluation of this parameter as a prognostic indicator.