The objective was to investigate the effect of variation in forage source and feed particle size of a diet, including interactions, on the amount and the composition of microbial crude protein (CP) in a semi-continuous culture system (Rusitec). Different microbial CP fractions were compared. Five diets with mean forage proportion of 0.88 and different maize silage to grass silage ratios (100 : 0, 79 : 21, 52 : 48, 24 : 76 and 0 : 100) were used. Diets were ground through sieves with a pore size of either 1 or 4 mm, matching the particle size of fine (F) and coarse (C), respectively. Diets were characterised by increasing concentrations of CP and fibre fractions, and decreasing concentrations of starch with ascending inclusion rates of grass silage. Microbial mass was isolated from feed residues after incubation from the liquid phase of the fermenter and from the liquid effluent. The amount of synthesised microbial CP was determined on the basis of 15N balance. It increased quite linearly by the stepwise replacement of maize silage by grass silage, and was higher in C treatments compared to F treatments. Efficiency of microbial CP synthesis (EMPS) was improved from 29 to 43 mg microbial N/g degraded organic matter (OM) by increasing the proportion of grass silage in the diet, but was unaffected by particle size. The N content as well as the profiles of amino acids of the three microbial fractions was affected by diet composition and particle size. The ratio of solid- to liquid-associated microbes was affected by diet composition and feed particle size. The amount and EMPS seemed to be improved by degradation of OM from grass silage and an increasing availability of N. Moreover, the results of this study indicated a shift in the composition of the microbial community caused by variation in forage composition and feed particle size.