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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 60 min, and with respect to protein concentration up to 750 μg ml−1. The optimum pH for enzyme activity was 8·5 using 10 mm-Tris/HCl buffer. Mg2+ was necessary for maximum activity and 10 mm-MgC12 was routinely used during the assays. The apparent K m for the substrate UDP-GlcNAc was 2 mm. Polyoxin D was a competitive inhibitor of chitin synthesis with an apparent K i of 4 μm. Following treatment with trypsin (12·5 μg ml−1), 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-KOH or 1 m-acetic acid, but it was solubilized by heating in 6 m-HCl at 120 °C for 2.5 h and was hydrolysed by chitinase into diacetylchitobiose.