The aim of this study was to evaluate the association between p53, p21, p27 and Rb expression, alone or in combination, with pathological features and clinical outcomes of urothelial carcinoma of the bladder. Immunohistochemical staining for p53, p21, p27 and pRB was performed on tissue microarrays comprising normal urothelium from nine controls, transurethral resection specimens from 74 patients with Ta, Tis and/or T1 bladder urothelial carcinoma, radical cystectomy specimens from 226 consecutive urothelial carcinoma patients, and lymph nodes with tumor invasion from 50 of the 226 cystectomy patients. All nine controls had normal status of biomarkers. The proportion of biomarkers alterations was highest in lymph node metastases. p53, pRB and p27 were associated with pathologic stage, lymphovascular invasion and lymph node metastases (P-values≤0.050). Each biomarker alone, each combination of two biomarkers and the number of altered biomarkers were all independently associated with disease recurrence and bladder cancer-specific death (P-values≤0.046). In patients with organ-confined disease, p53, p21 and p27 were independently associated with disease recurrence and bladder cancer-specific death (P-values≤0.046). There was a good but not perfect concordance in the expression of biomarkers between matched transurethral resection and cystectomy specimens (n=22; concordance rates: 77–86%) and between matched lymph node and cystectomy specimens (n=50, 70–92%). In conclusion, p53, p21, p27 and pRB have a cooperative/synergistic role in the biologic behavior of bladder urothelial carcinoma. In contrast to the use of single biological markers, evaluation of their combined status may improve prognostic models and help identify patients who might benefit from adjuvant therapies and in decision-making.