Chitin synthase activity was detected in actively growing mycelium of Neocallimastix frontalis after mechanical disruption of the cells. Chitin formation in fungal extracts at 32 "C was linear with respect to time for at least 60min, and with respect to protein concentration up to 750 pg ml-l. The optimum pH for enzyme activity was 8.5 using 10 mM-Tris/HCl buffer. Mg2+ was necessary for maximum activity and 10 mM-MgC1, was routinely used during the assays. The apparent K, for the substrate UDP-GlcNAc was 2 m ~P.o lyoxin D was a competitive inhibitor of chitin synthesis with an apparent Ki of 4 p ~F.o llowing treatment with trypsin (12.5 pg ml-I ), the chitin synthase activity of the fungal extract increased by six-fold, indicating that most of the chitin synthase activity was zymogenic. The reaction product was insoluble in 1 M-KOHo r 1 M-acetic acid, but it was solubilized by heating in 6 M-HCa~t 120 "C for 2.5 h and was hydrolysed by chitinase into diacetylchitobiose.