The main purpose of this retrospective study was to compare Ki-67 expression in operable breast cancer examined by immunostaining and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Relations between Ki-67 and classic prognostic factors were also investigated, and the prognostic relevance of Ki-67 expression was examined. Expression of Ki-67 was analyzed in specimens of invasive ductal breast cancer tissue obtained from 131 women during radical mastectomy. There was a significant, but weakly positive, correlation between Ki-67 expression assessed by immunostaining and real-time RT-PCR (tau=0.154, p=0.005). Higher Ki-67 expression in immunostaining and RT-PCR was more often seen in grade 3 tumors (p<0.001 and p=0.026, respectively). No significant relationship with age, disease stage, nodal involvement, estrogen receptor or HER2 status was found. In a univariate and multivariate analysis of cancer-specific survival with a median follow-up of 56 months, Ki-67 expression determined by immunostaining or real-time RT-PCR was no prognostic factor. We demonstrated that Ki-67 expression levels measured by immunostaining and real-time RT-PCR were weakly concordant, and both were related to higher tumor grade. Ki-67 expression did not influence survival.