BACKGROUND Unilateral ablative strategy success depends on reliable prediction of prostate cancer (PCA) location. We evaluated the discrepancy in PCA localization between unilateral positive biopsy (PBx) and radical prostatectomy (RP). METHODS Between 2004 and 2008, 431 patients were diagnosed with unilateral PCA by 12-core PBx; 179 underwent RP and constituted our study cohort. Specimens were reviewed to map tumor outline and determine number of cancer foci, tumor volume, Gleason score (GS), zone of origin, localization, and pathologic stage. RESULTS In 50 men, biopsy and prostatectomy findings correlated (unilateral tumor); in 129, PCA was detected in the contralateral side of the prostate. In 52 patients, 54 clinically significant tumors were missed by biopsy. When patients with true unilateral and missed contralateral disease at RP were compared with respect to prognostic parameters no significant differences were detected. Sixty-one of the 88 patients with preoperative low-risk disease had true unilateral (n = 21) or missed insignificant contralateral (n = 40) PCA; 27 had missed significant contralateral PCA at RP. PSA > 4 ng/ml predicted presence of significant bilateral disease in low-risk population (P = 0.004). Twenty-four of 27 patients with significant bilateral cancer had PSA > 4, although 33/61 with unilateral or bilateral insignificant cancer had similar elevated PSA values. CONCLUSIONS Twelve-core biopsy is inadequate to identify candidates for organ-sparing therapy. Most men with unilateral positive biopsies have bilateral cancer at prostatectomy. Tumors missed by biopsy were clinically significant in 40% of patients, but no prognostic parameters could predict unilateral disease. Hemi-ablative treatment might fail to eradicate significant lesions in the contralateral side.