AbstractLateral gene transfer (LGT) into multicellular eukaryotes with differentiated tissues, particularly gonads, continues to be met with skepticism by many prominent evolutionary and genomic biologists. A detailed examination of 26 animal genomes identifed putative LGTs in invertebrate and vertebrate genomes, concluding that there are fewer predicted LGTs in vertebrates/chordates than invertebrates, but there is still evidence of LGT into chordates, including humans. More recently, a reanalysis a subset of these putative LGTs into vertebrates concluded that there is not horizontal gene transfer in the human genome. One of the genes in dispute is an N-acyl-aromatic-L-amino acid amidohydrolase (ENSG00000132744), which encodes ACY3, which was initially identified as a putative bacteria-chordate LGT but was later debunked has a significant BLAST match to a more recently deposited genome of Saccoglossus kowalevskii, a flatworm, Metazoan, and hemichordate. Using BLAST searches, HMM searches, and phylogenetics to better understand the evidence for lateral gene transfer, gene loss, and rate variation in ACY3/ASPA homologues, the most parsimonious explanation for the distribution of ACY3/ASPA genes in eukaryotes likely involves both gene loss and lateral gene transfer, albeit lateral gene transfer that occurred hundreds of millions of years ago prior to the divergence of gnathostomes and even longer and prior to the divergence of bilateria. Given the many known, well-characterized, and adaptive lateral gene transfers from bacteria to insects and nematodes, lateral gene transfers at these time scales in the ancestors of humans is expected.