The stromal microenvironment has key roles in prostate development and cancer, and cancer-associated fibroblasts (CAFs) stimulate tumourigenesis via several mechanisms including the expression of pro-tumourigenic factors. Mesenchyme (embryonic stroma) controls prostate organogenesis, and in some circumstances can re-differentiate prostate tumours. We have applied next-generation Tag profiling to fetal human prostate, normal human prostate fibroblasts (NPFs) and CAFs to identify molecules expressed in prostatic stroma. Comparison of gene expression profiles of a patient-matched pair of NPFs vs CAFs identified 671 transcripts that were enriched in CAFs and 356 transcripts whose levels were decreased, relative to NPFs. Gene ontology analysis revealed that CAF-enriched transcripts were associated with prostate morphogenesis and CAF-depleted transcripts were associated with cell cycle. We selected mRNAs to follow-up by comparison of our data sets with published prostate cancer fibroblast microarray profiles as well as by focusing on transcripts encoding secreted and peripheral membrane proteins, as well as mesenchymal transcripts identified in a previous study from our group. We confirmed differential transcript expression between CAFs and NPFs using QrtPCR, and defined protein localization using immunohistochemistry in fetal prostate, adult prostate and prostate cancer. We demonstrated that ASPN, CAV1, CFH, CTSK, DCN, FBLN1, FHL1, FN, NKTR, OGN, PARVA, S100A6, SPARC, STC1 and ZEB1 proteins showed specific and varied expression patterns in fetal human prostate and in prostate cancer. Colocalization studies suggested that some stromally expressed molecules were also expressed in subsets of tumour epithelia, indicating that they may be novel markers of EMT. Additionally, two molecules (ASPN and STC1) marked overlapping and distinct subregions of stroma associated with tumour epithelia and may represent new CAF markers.