To fill this gap, we herein report on the characterization of a representative member of the family, Archaeoglobus fulgidus G3PS (AfG3PS), and describe methods to facilitate the analytical entry into research with these enzymes. Like all G3PSs, AfG3PS has previously been sparsely characterized, with only two studies describing this enzyme. The Pai group reported two crystal structures of recombinant AfG3PS and the Babinger group qualitatively confirmed its activity in vivo. We found that AfG3PS shares over 50% sequence identity with at least 80 other family members and family-wide sampling of the structural space with ColabFold (Fig. ) indicated that AfG3PS would be a good model system to represent G3PS active sites and, therefore, their mechanism. Thus, we used high-throughput kinetics with substrate analogues, spectroscopy, biophysical and computational methods to provide the first in-depth biocatalytic and mechanistic forays into this enzyme family. We found that AfG3PS is a highly promiscuous enzyme, capable of making various (E)-and (Z)-configured prenyl glycerol ethers while exerting perfect control over the configuration at the glycerol unit. We also present evidence in support of a mechanism involving an intermediary prenyl carbocation equivalent formed through recruitment of, and catalysis by, two mechanistically essential magnesium ions. As such, our study provides the fundamental understanding and methods to introduce G3PSs into the biocatalytic alkylation toolbox.