The effect of different water availabilities (water activity, aw; 0.98-0.93) and time (up to 15 days) on the production of seven hydrolytic enzymes by strains of F. moniliforme and F. proliferatum during early colonisation of gamma-irradiated living maize grain were examined in this study. Both the total activity (micromol 4-nitrophenol min(-1) g(-1) maize) and specific activity (nmol 4-nitrophenol min(-1) microg(-1) protein) were quantified using chromogenic p-nitrophenyl substrates. The dominant three enzymes produced by the fungi on whole colonised maize kernels were alpha-D-galactosidase, beta-D-glucosidase, and N-acetyl-beta-D-glucosaminidase. The other four enzymes were all produced in much lower total amounts and in terms of specific activity (beta-D-fucosidase, alpha-D-mannosidase, beta-D-xylosidase and N-acetyl-alpha-D-glucosaminidase), similar to that in uncolonised control maize grain. There were significant increases in the total production of the three predominant enzymes between 3-15 days colonisation, and between 3-6 days in terms of specific activity when compared to untreated controls. The total and specific activity of the alpha-D-galactosidase, beta-D-glucosidase and N-acetyl-beta-D-glucosaminidase, were maximum at 0.98 aw with significantly less being produced at 0.95 and 0.93 aw, with the exception of the total activity of alpha-D-galactosidase which was similar at both 0.95 and 0.93 aw. Single factors (time, aw, and inoculation treatment), two- and three- way interactions were all statistically significant for the three dominant enzymes produced except for specific activity of beta-D-glucosidase (two and three-way interactions) and for total activity of alpha-D-galactosidase in the time x aw treatment. This study suggests that these hydrolytic enzymes may play an important role in enabling these important fumonisin-producing Fusarium spp. to rapidly infect living maize grain over a wide aw range.