Multiplex mRNA profiling by a reverse transcription-polymerase chain reaction (RT-PCR) has been reported in the last few years as a new approach for the identification of body fluids. We have also demonstrated the feasibility of identifying body fluids by using a real-time RT-PCR assay. Statherin (STATH) and histatin (HTN3), the selected genes for saliva, and protamin 2 (PRM2) and semenogelin 1 (SEMG1), those selected for semen, showed high specificity to these body fluids. Thus, the sensitivity and specificity of target genes were examined in body fluid stains. All target genes were detected in 0.1 microL 6-day-old stains, and showed high specificity in 7-day-old 30 microL stains. Furthermore, the stability of HTN3 in saliva stains was examined under various environmental conditions over time. The results showed that the degradation of mRNA in the stains was highly affected by wet conditions, and that light was also an important factor. However, mRNA was detectable in an older saliva stain (6 years old) and in an older semen stain (3.5 years old), both of which had been kept under dry and dark conditions. The stability of mRNA beyond our supposition may play an important role in developing new techniques for body fluid identification.