Cholinesterases (ChEs) are highly polymorphic proteins, capable of rapidly hydrolyzing the neurotransmitter acetylcholine and involved in terminating neurotransmission in neuromuscular junctions and cholinergic synapses. In an attempt to delineate the structure and detailed properties of the human protein(s) and the gene(s) coding for the acetylcholine hydrolyzing enzymes, a human cDNA coding for ChE was isolated by use of oligodeoxynucleotide screening of cDNA libraries. For this purpose, a method for increasing the effectiveness of oligonucleotide screening by introducing deoxyinosine in sites of codon ambiguity and using tetramethyl-ammonium salt washes to remove false-positive hybrids was employed. The resulting isolated 2.4-kilobase (kb) cholinesterase cDNA sequences encode for the entire mature secretory protein, preceded by an N-terminal signal peptide. The human ChE primary sequence shows almost no homology to other serine hydrolases, with the exception of a hexapeptide at the active site. In contrast, it displays extensive homology with acetylcholinesterase form Torpedo californica and Drosophila melanogaster as well as with bovine thyroglobulin. These extensive homologies probably suggest the need of the entire coding sequence for the physiological function(s) fulfilled by the enzyme and further suggest a common, unique, ancestral gene for these cDNAs. In turn, the cDNA was used as a probe to isolate genomic DNA sequences for the 5'-region of the human ChE gene. The genomic DNA fragment encoding part of the 5'-region of ChEcDNA was detected by DNA blot hybridization, enriched 70-fold by gel electrophoresis and electroelution, cloned in lambda phage and isolated. Sequencing of the cloned DNA revealed that it did indeed include part of the 5'-region of ChEcDNA, starting at an adjacent 5'-position to the nucleotides coding for the initiator methionine, and ending with an EcoRI restriction site inherent to the ChEcDNA sequence. The isolated fragment of the human cholinesterase gene is currently employed to complete the structural characterization of this and related genes.