Tissue microarrays were constructed using prostate needle biopsy specimens obtained from 58 patients who underwent radical prostatectomy for localized or locally advanced prostate cancer (PC). We used the spiral array (SA) technique, a novel approach for tissue array construction in a spiral form, which has advantages over small needle biopsy specimens. Roll-shaped tissue pieces produced by slicing a prostate biopsy tissue block and trimming the cancer segment were used to obtain a tissue array block. Cancer segments measuring >3 mm were incorporated into the tissue arrays. Cancer fragments (n=253) were obtained from formalin-fixed, paraffin-embedded needle biopsy specimens. The median number of cancer fragments per patient was four (1-8, min-max). On SA, the median number of confirmed cancer fragments per patient was four (1-7) and 224 cancer fragments (88.5%) were confirmed histologically. Each core of reeled tissue contained at least one cancer fragment. The expressions of multiple prognostic molecular markers for PC (Ki-67, p53 and bcl-2) were immunohistochemically measured using the SA. The Ki-67 and bcl-2 expressions were significantly associated with the Gleason score (GS). A univariate analysis identified Ki-67, bcl-2 and GS as significant predictors of cancer-specific survival, p53 and bcl-2 as significant predictors of overall survival and Ki-67, adjuvant androgen deprivation and GS as significant predictors of biochemical progression. In a multivariate analysis, p53 was independently associated with overall survival, while adjuvant androgen deprivation and GS were associated with biochemical progression. These results indicate that SA has potential as a new tool for translational research on PC.