This paper presents radiochemical micro-assays for the determination of choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) activities. The methods rely on the reaction between labeled acetyl-CoA and unlabelled choline to form labeled acetylcholine, which is then isolated using various extraction techniques such as precipitation with sodium tetraphenylboron or extraction with ketonic sodium tetraphenylboron. The assays were tested on samples from central and peripheral nervous tissues and purified enzymes. The blank values for ChAc and AChE correspond to 20 ng and 5 ng of brain tissue, respectively. The sensitivity and advantages of the methods over existing techniques are discussed, including the use of synthetic acetyl-CoA and acetyl-CoA formed in situ. The methods were found to be reproducible and provided good agreement between different assay procedures. The blank values were independent of the tissue studied and were lower than those reported in other methods. The use of synthetic acetyl-CoA in the ChAc assay was found to yield slightly lower values, possibly due to an inhibitory contaminant in some acetyl-CoA preparations. The AChE assay method was found to be comparable in sensitivity to other recently published methods.This paper presents radiochemical micro-assays for the determination of choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) activities. The methods rely on the reaction between labeled acetyl-CoA and unlabelled choline to form labeled acetylcholine, which is then isolated using various extraction techniques such as precipitation with sodium tetraphenylboron or extraction with ketonic sodium tetraphenylboron. The assays were tested on samples from central and peripheral nervous tissues and purified enzymes. The blank values for ChAc and AChE correspond to 20 ng and 5 ng of brain tissue, respectively. The sensitivity and advantages of the methods over existing techniques are discussed, including the use of synthetic acetyl-CoA and acetyl-CoA formed in situ. The methods were found to be reproducible and provided good agreement between different assay procedures. The blank values were independent of the tissue studied and were lower than those reported in other methods. The use of synthetic acetyl-CoA in the ChAc assay was found to yield slightly lower values, possibly due to an inhibitory contaminant in some acetyl-CoA preparations. The AChE assay method was found to be comparable in sensitivity to other recently published methods.