Oligodeoxynucleotide primers, based on amino acid sequences conserved in known acetylcholinesterases (AChEs), were used in reverse-transcription polymerase chain reaction (RT-PCR) with mRNA from Boophilus microplus (Canestrini) as the template. Primer walking and rapid amplification of cDNA ends (RACE) techniques were used to complete the cDNA sequence identified by RT-PCR. The complete B. microplus cDNA sequence contained an open reading frame encoding a 620 amino acid protein with a 20 amino acid signal peptide at the N-terminus targeting the protein for the secretion pathway. BLAST searches of GenBank using the presumptively encoded protein revealed highest sequence similarity to AChEs. The presumptively encoded protein was of similar size and structural properties to other identified AChEs, including the presence of the catalytic triad (Ser, Glu, His) and appropriate placement of internal cysteines to yield three internal disulfide bonds corresponding to those of known AChEs. Putative conserved domains identified the sequence as a member of the carboxylesterase family, pfam00135.8, of which AChE is a member. This cDNA therefore presumptively encodes a third transcribed AChE (AChE3) cDNA of B. microplus. Comparison of the three AChE cDNA sequences expressed in B. microplus demonstrated no discernible nucleotide sequence homology and relatively low amino acid sequence homology, strongly suggesting that they are not alleles of one another. The potential presence of multiple expressed AChEs in B. microplus suggests alternative mechanisms for development of resistance to pesticides that target AChE. The homology-based identification of a third expressed AChE in B. microplus is a surprising result and strongly implies the need for confirmation of gene identity for presumptive AChEs.