A dual flow continuous culture system was used to examine the effect of replacing plant protein-N with urea-N plus maize in diets containing predominantly alkaline hydrogen perioxide-treated wheat straw (AHP-S) on bacterial protein synthesis and other fermentation measurements. Four diets consisted of 70% AHP-S with urea-N replacing 0, 25, 50 and 75% of soya-bean meal (SBM)-N. To maintain isonitrogenous diets and equal levels of AHP-S, maize was added with increasing levels of urea. Each diet was replicated four times. Data were tested for linear, quadratic and cubic effects using orthogonal contrasts. No effects (P > 0.05) due to treatment were observed for true dry matter or organic matter digestion. However, a trend towards greater digestion as urea-N increased was noted, probably because of the increased level of maize in the diet. Ammonia concentration showed a linear (P < 0.01) and cubic (P < 0.05) response to increasing urea-N levels. The cubic effect was probably due to the increasing amounts of maize in diets where urea-N increased. Non-ammonia-N flow decreased linearly (P < 0.01) with increasing urea-N levels, resulting from decreasing dietary SBM protein. Increasing levels of urea-N elicited a cubic response (P < 0.05) in bacterial-N flow, possibly because of associative effects resulting from increasing proportions of maize when urea-N replaced SBM-N in the diets. Digestion of structural carbohydrates was not affected (P > 0.05) by substitution of plant protein-N with urea-N. In contrast, total non-structural carbohydrate digestion increased linearly (P < 0.01) with increasing levels of urea-N plus maize in the diet. Urea-N plus maize did not affect (P > 0.05) total volatile fatty acid concentration in the effluent; however, molar proportions of isobutyrate and isovalerate decreased linearly (P < 0.01) with increasing amounts of urea-N plus maize in the diet. Results of this experiment indicated that replacement of up to 25% of the supplemental plant protein-N with urea-N plus maize did not have a negative effect on ruminal bacterial growth and fermentation in continuous culture.