Glycine conjugation is an important phase II reaction and represents a central detoxification pathway which is essential for the recycling of free coenzyme A. Only few sequence variants have been reported in the human GLYAT gene and only two studies have overexpressed the human protein in bacterial systems and partially characterized it. This has prompted us to study the wild-type enzyme and two sequence variants not only in the E.?coli strain Origami 2(DE3), but also to overexpress GLYAT in HEK293?cells, a human-derived cell line. Following purification of the recombinant proteins from E.?coli the wild-type GLYAT protein and sequence variants, p.(Gln61Leu) yielded decreased specific activity than the wild-type enzyme, while specific activity of p.(Asn156Ser) activity of the latter variant was somewhat increased. K values were similar for the three forms of GLYAT overexpressed in bacteria and for the wild-type enzyme overexpressed in HEK293?cells. Localization studies demonstrated intramitochondrial localization of human wild-type GLYAT, conjugated with eGFP, in the HEK293?cells. As p.(Gln61Leu) does not only impair GLYAT activity in?vitro, but is of high prevalence in a Caucasian Afrikaner cohort in South Africa, potential pharmacogenetic implications, warrant further studies of GLYAT.