Purpose: Alterations of the androgen receptor (AR)-mediated signaling through numerous mechanisms are increasingly recognized in prostate cancer (CaP) progression. We hypothesized that the assessment of well-defined AR transcriptional targets (e.g., PSA/HK3 mRNA) in CaP tissues will provide in vivo readout of AR dysfunctions. Moreover, quantitative expression features of PSA/HK3 mRNA in prostate tumor cells may serve as a prognostic indicator of disease progression. Experimental Design: Paired benign and malignant epithelial cells (242 specimens) were obtained from laser capture microdissection of frozen OCT-embedded tissue sections prepared from radical prostatectomy specimens of 121 patients. Quantitative expression of PSA/HK3 mRNA in the matched malignant and benign cells was analyzed by real-time reverse transcription-PCR. Results: CaP cells express significantly lower PSA/HK3 mRNA levels than matched benign cells (P = 0.0133). Moreover, low PSA/HK3 mRNA expression inmalignant cells was associated with increased risk of biochemical recurrence (P = 0.0217), as well as with time to recurrence (P = 0.0371), in patients with intermediate preoperative serum prostate-specific antigen levels (2-10 ng/mL). The expression of androgen-dependent genes in clinical samples correlates with each other in patients with higher expression of PSA/HK3 mRNA but not in patients with lower expression of PSA/HK3 mRNA reflecting AR pathway dysfunction. Conclusions: Our study has unraveled a novel prognostic utility of quantitative measurements of PSA/HK3 mRNA reflecting AR transcriptional activity in CaP cells, which is independent of serum prostate-specific antigen. It also has potential in stratifying subsets of patients exhibiting progressive disease associated with dampened AR transcriptional functions who may be targeted by tailored therapeutic strategies.