BACKGROUND Almost all attempts at establishing prostate carcinoma cell lines have resulted in generation of cells that are androgen-independent, including commonly used LNCaP which expresses androgen receptor (AR) and AR-negative Du145 and PC-3. We attempted to clarify the mechanism(s) responsible for the failure to respond to androgen. METHODS Cell lines LNCaP, CWR22R, PC-3, Du145, and CA7T2CL were used to examine the AR promoter function with a reporter gene assay and its methylation status by Southern blot, PCR of bisulfite-converted DNA, and 5-aza-2'-deoxycytidine treatment. Structural abnormalities of the AR were identified by sequencing of reverse-transcribed mRNA. RESULTS All tested AR-positive prostate carcinoma cells were capable of AR transcription at a significantly higher level than PC-3 and Du145, thus suggesting relative deficiency of the transcription factors in the AR-negative cells, further associated with methylation. Examination of CWR22R cells, which express the AR but are androgen-independent, identified an in-frame duplication of exon 3, which resulted in insertion of 39 amino acids in the DNA-binding domain. CONCLUSIONS Relative deficiency of transcription factors associated with methylation is responsible for the lack of AR promoter function in most of AR-negative cell lines. Mutations in the AR gene are present in the cells that express the AR but are androgen-independent.