Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall of Giardia and is synthesized by inducible enzymes in the cytosol of encysting trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encysting Giardia is reported, and this activity exclusively incorporates [1-(14)C]GalNAc (from UDP-[(14)C]GalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall of GIARDIA: This ethanol precipitate exhibits characteristics of Giardia cyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS). CWS activity peaks in cells between 24 and 36 h of encystment and exhibits a high affinity and marked specificity for UDP-GalNAc as its substrate. UDP-N-acetylglucosamine, UDP-glucose, UDP-galactose, D-glucosamine and D-galactosamine were not incorporated into the ethanol precipitate. Partially purified CWS activity exhibits an apparent K(m) of 0.048 mM for UDP-GalNAc, a V(max) of 0.70 nmol x min(-1) (mg protein)(-1) and a requirement for divalent cations in the following order of preference: Ca(2+), Mg(2+)>Co(2+)>>>Mn(2+), Zn(2+). EDTA inhibits CWS activity.