Extensive scientific literature data point to reciprocal interactions between prostate stromal cells and prostate cancer cells that likely regulate tumor progression. To investigate whether these intratumoral-reactive stromal cells in human prostate cancer are predictive of survival, tumor stroma volume and specific stroma markers were quantitated by using tissue microarrays (index tumors of 847 patients), and the results were analyzed relative to the recurrence-free survival data set for these patients. Tumor tissue was evaluated with Masson's trichrome stains and by immunohistochemistry with antibody probes to smooth muscle alpha-actin, desmin, vimentin, pro-collagen type I, and calponin. The relative volume of intratumor stroma (5% stroma, grade 0; 5-15%, grade 1; 15-50%, grade 2; >50%, grade 3) and the expression index of stromal marker (staining intensity grade x percentage of positive cells per field) were quantitated and analyzed. Interpretable data were obtained from 545 patients. Statistical analysis of the survival data set showed that the volume of reactive stroma in the tumor was a significant predictor of disease-free survival. Stroma volume was most optimal as an independent predictor in tumors containing stroma, defined as Gleason 7 and lower grades. Of interest, tumors with either little to no stroma or tumors with abundant stroma each showed reduced recurrence-free survival. For specific stromal markers, reduced desmin and smooth muscle alpha-actin were hallmarks of cancer-associated reactive stroma relative to normal fibromuscular stroma. Quantitative analysis of desmin and smooth muscle alpha-actin expression showed both to be significant and independent predictors of recurrence-free survival. This is the first study to demonstrate that nonepithelial-reactive stroma elements in prostate cancer tumors can be used as prognostic indicators. These data also add to the concept that tumors are not purely epithelial and the tumor-reactive stroma must be considered an important biological component of the cancer.