In order to examine the molecular basis for regional variation in expression of brain acetylcholinesterase (AChE), an assay using reverse transcription and polymerase chain reaction (RT-PCR) was developed to measure steady state levels of AChE mRNA. The amplification method was designed to be specific for templates derived from AChE mRNA and to avoid potential artifacts induced by the presence of genomic DNA. RT-PCR made it possible to assay AChE mRNA in milligram samples from different regions of the rat brain. Determinations by RT-PCR were faster and more sensitive than Northern blotting. The results, including a surprisingly low level of AChE mRNA in the caudate nucleus, agreed with earlier observations by Northern blot and in-situ hybridization. Quantitative RT-PCR may be useful in future studies on developmental and physiological regulation of AChE expression in the brain.