The objective of this study was to determine whether the overexpression of bcl-2, a key protein governing the apoptotic response to radiation, adds to pRb status in estimating the propensity for radiation response in patients with muscle-invasive bladder cancer. Archival formalin-fixed, paraffin-embedded, pretreatment bladder tumor samples were available in 109 of 301 patients treated preoperatively with 50 Gy in 25 fractions followed by radical cystectomy 4-6 weeks later. Radiation response was assessed by clinical-to-pathological tumor downstaging or upstaging. Altered expression of bcl-2 (47% of 107 patients), p53 (56% of 109 patients), and pRb (30% of 98 patients) was assessed by immunohistochemical staining. Morphological criteria were used to calculate the percentage of apoptotic cells. bcl-2 staining correlated with tumor grade; all grade 2 tumors (n = 7) displayed normal bcl-2 expression (negative staining). No correlations between bcl-2 staining and pretreatment apoptosis levels, p53 staining, and pRb staining were observed. In terms of the radiation response parameters, univariate analyses revealed that bcl-2 overexpression was the only factor associated with upstaging. The main predictor of downstaging was the loss of pRb expression (negative staining). Multivariate logistic regression confirmed these findings and also showed that normal pRb expression (positive staining) was significantly related to upstaging. Patient outcome was adversely affected by bcl-2 overexpression, because these patients experienced significantly increased actuarial local failure rates. No difference in distant metastasis or survival rates by bcl-2 staining was seen. The strongest independent correlates of radiation response thus far identified in muscle-invasive bladder cancer are from bcl-2 and pRb immunohistochemical staining. The overexpression of bcl-2 and the normal expression of pRb seem to thwart the apoptotic response to radiation via independent mechanisms. Abnormalities in the expression of proteins that regulate apoptosis may prove to establish a molecular phenotype to characterize which patients should receive radiotherapy.