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The basal cell-specific cytokeratin antibody (34betaE12) is widely used to aid in the diagnosis of cancer in challenging prostate needle biopsies (NBX) and transurethral resections of the prostate (TURP). Because prostate carcinoma (PCa) lacks basal cells, the absence of basal cell as determined by 34betaE12 can aid in the confirmation of a histologically suspicious lesion. However, false-negative staining occurs because of patchy cytoplasmic staining, making a definitive diagnosis difficult. A recently identified basal cell marker p63, a p53 homologue, stains basal cell nuclei but not secretory cells. The aim of this study is to determine if the p63 antibody offers any clinically useful advantage over 34betaE12 in the diagnosis of challenging atypical prostate lesions. Ninety-four cases, comprised of 25 consecutive prostate NBX and 2 TURP with an atypical suspicious focus, 55 NBX cases of histologically unequivocal PCa and 12 TURP specimen removed for benign prostate hyperplasia, were stained with the monoclonal antibodies 34betaE12 and 4A4 anti-p63. Basal cell staining intensity, percentage basal cell-positive glands in benign, malignant, and atypical foci, and number of benign glands not staining were evaluated for 34betaE12 and p63 stains. A total of 67 prostate NBX cases, including one TURP, were diagnosed with PCa, 1 atypical small acinar proliferation, 10 benign, and 4 cases excluded because of lost tissue on step sections. None of the 67 PCa NBX cases demonstrated 34betaE12 or p63 immunoreactivity (100% specific). Whereas 57 of 108 (53%) prostate NBX cores from 78 cases demonstrated a similar percentage of basal cell staining for both antibodies, 45 of 108 (41%) NBX cores demonstrated a higher percentage of p63 basal cell staining in benign glands. Only 6 of 108 NBX (6%) cores had a higher percentage of basal cell staining with 34betaE12 (Wilcoxon signed rank test, p <0.0001). Lack of basal cell staining in more than two benign glands occurred in 25 of 108 (23%) and 10 of 108 (9%) prostate NBX cores stained with 34betaE12 and p63, respectively. In the vast majority of atypical cases, both 34betaE12 and p63 staining differences were not clinically significant, except in 2 of 27 (7%) cases p63 offered diagnostic utility beyond the 34betaE12 immunostain. p63 in these cases demonstrated discontinuous but strong staining in atypical glands and adjacent benign glands, whereas 34betaE12 failed to stain optimally in this critical area. For 12 TURP cases the mean percentage basal cell positivity in benign glands was 75% and 95% for 34betaE12 and p63, respectively (p = 0.006). Lack of basal cell staining in more than two glands occurred in 12 of 12 (100%) and 2 of 12 (17%) TURP specimens stained with 34betaE12 and p63, respectively (p <0.0001). In summary, 34betaE12 and p63 are highly specific for basal cells and therefore are negative in areas of PCa. p63 is more sensitive than 34betaE12 in staining benign basal cells, particularly for TURP specimens, offering slight advantage over 34betaE12 in diagnostically challenging cases. p63 may be used as an alternative to 34betaE12 stain for difficult prostate lesions.