Prostate cancer originates in the outer gland in 80% of patients and in the transition zone (TZ) in 20%. Transition zone lesions have historically been the most difficult to identify. There have been discrepancies regarding the importance of routinely performing biopsies of the inner gland when performing biopsies of the outer gland. To determine how often TZ tumors were diagnosed as the sole area of cancer, and to determine how frequently TZ tumors contained higher-grade cancer than the outer gland, we attempted to assess the value of including the TZ in routine biopsies of the prostate when no lesions are seen by gray-scale ultrasound (US). A retrospective review of 619 consecutive ultrasound-guided biopsies divided the subjects into: 1) directed biopsies of specific US-identified lesions (N = 140); 2) directed biopsies of specific US-identified lesions and quadrants (N = 165) or sextants (N = 174), including the TZ; and 3) quadrant (N = 46) or sextant (N = 93) biopsies without a focal US-identified lesion. Overall, 185 patients (29.9%) with cancer were identified. Of these, 21.4% (N = 30) of men with US-identified, focal lesion biopsies alone had cancer. Of those men with cancer, 35.8% (N = 59) with US-identified lesions and quadrant biopsies had cancer; 37.4% (N = 65) of those with focal lesions and sextant biopsies had cancer; 23.9% (N = 11) of men without focal lesions but quadrant biopsies had cancer; 21.5% (N = 20) of men without focal lesions but with sextant biopsies had cancer. A total of 267 sextant biopsies were performed: 3.0% (N = 8) of these patients were found to have cancer in the TZ alone without involvement of the outer gland; 12.4% (N = 33) had TZ and outer gland cancers, of which 18.2% (N = 6) had a higher Gleason grade cancer in the TZ than in the outer gland. Routine sextant biopsies in lieu of quadrant biopsies, including the TZ, for men with or without focal lesions will yield a small increase in the number of prostate cancers diagnosed or may identify a higher grade of cancer than would be expected from using quadrant biopsies alone.