The aim of this study was to investigate the expression of the muscarinic acetylcholine receptor (mAchR) subtypes mRNA in primary culture of human prostate stromal cells using the reverse transcription polymerase chain reaction (RT-PCR), RNA blotting and in situ hybridization (ISH). Using an explant method, we obtained a primary culture of prostate stromal cells from three patients with benign prostatic hypertrophy. Total RNA was extracted using the acid guanidinium method for cDNA synthesis. First-strand cDNA was then used for PCR with primers designed to amplify the fragments of each mAchR subtypes (m1-m5) cDNA sequence. The m2, m3 and m4 subtype expected bands were detected; in particular m2 transcripts was strongly detected in the stromal cell culture. Each of the PCR products were subcloned into the pGEM-T plasmid vector, sequenced and random primer labeled using 32P. Digoxigenin-labeled cRNA probes were synthesized by in vitro transcription. RNA blotting using a m2 muscarinic receptor cDNA probe revealed a 4.5 kb single transcript. However, m3 and m4 probes did not hybridize. Using in situ hybridization (ISH), m2 receptor mRNA signals were detected in several smooth muscle cells. The staining was predominantly localized to the perinuclear cytoplasm. The m3 and m4 probes did not hybridize. These results suggested that m2 receptor subtype plays a role in smooth muscle activity of the human prostate.