The majority of the human genome is transcribed as non-protein-coding RNAs (ncRNAs) derived mainly from intergenic and intronic regions, in many cases transcribed from both DNA strands, generating sense and antisense RNAs. ncRNAs have emerged as an important class of regulatory molecules in eukaryotes, but still remain largely uncharacterized. Elucidating the mechanisms of biosynthesis of ncRNAs is essential to better understand this class of transcripts. Based on the observation that many long ncRNAs share structural features with mRNAs, it has been widely assumed these ncRNAs are transcribed by RNA Polymerase II (RNAP II). However, recent data suggest that RNAP II cannot solely account for the expression of all non-annotated transcripts observed in humans. In order to evaluate the participation of RNAP II in the synthesis of ncRNAs, we blocked RNAP II transcription in LNCaP, a human prostate cancer cell line, with the specific inhibitor α-amanitin (50μg/mL for 24h), and compared the expression of treated to non-treated cells using a 4,608-elements cDNA microarray enriched in intronic sequences. As expected, all prostate markers and housekeeping genes were down-regulated following treatment. The majority (41/53) of differentially expressed protein-coding genes were significantly down-regulated, while 12 genes were up-regulated upon RNAP II inhibition. In contrast, most intronic transcripts (20/25) had their expression increased, indicating the involvement of a distinct transcriptional mechanism. To further investigate this hypothesis, an oligoarray containing ~42,000 features, designed after extensive bioinformatics selection, was used to assess the effect of RNAP II inhibition in LNCaP cells. Again, only a small subset (25/1458) of protein-coding exon transcription was stimulated (two-fold or more) in treated cells. Moreover, the majority of intronic transcripts affected by α-amanitin were up-regulated, representing 66% (29/44) and 78% (47/60) of the intronic antisense and sense transcripts, respectively. Recently, a forth nuclear RNA Pol, named single-polypeptide RNAP IV (spRNAP IV), was reported transcribing a few mRNAs in human cells. Surprisingly, its activity is stimulated after α-amanitin treatment. Considering our observation that many intronic ncRNAs do not seem to be transcribed by RNAP II in human cells, it is possible that spRNAP IV or another unknown RNAP might be responsible for biosynthesis of this class of ncRNAs.