Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.