The teicoplanin acyltransferase (Atf) responsible for N-acylation of the glucosamine moiety to create the teicoplanin lipoglycopeptide scaffold has recently been identified. Here we use that enzyme (tAtf) and the cognate acyltransferase from the related A-40,926 biosynthetic cluster (aAtf) to evaluate specificity for glycopeptide scaffolds and for the acyl-CoA donor. In addition to acylation of 2-aminoglucosyl glycopeptide scaffolds with k cat values of 400–2000 min −1 , both Atfs transfer acyl groups to regioisomeric 6-aminoglucosyl scaffolds and to glucosyl scaffolds at rates of 0.2–0.5 min −1 to create variant lipoglycopeptides. Using the teicoplanin glycosyltransferase tGtfA, tAtf, and GtfD, a glycosyltransferase from the vancomycin producer, it is possible to assemble a novel lipoglycopeptide with GlcNAc at β-OH-Tyr 6 and an N 6 -acyl-glucosaminyl-vancosamine at Phegly 4 . This study illustrates the utility of chemo- and regioselective acyltransferases and glycosyltransferases to create novel lipoglycopeptides.