Five ruminally, duodenally, and ileally cannulated sheep (average BW 62 kg) were fed 65% roughage: 35% concentrate diets (CP = 15%) in a 5 x 5 Latin square design to study the applicability of using a combination of defaunation with N supplements (soybean meal [SBM], corn gluten meal [CGM], blood meal [BM], urea, and casein) with different extents of ruminal degradation to manipulate microbial protein synthesis and amount of ruminal escape protein. Diets were fed twice daily (1,759 g DM/d). Defaunation was accomplished with 30-ml doses of alkanate 3SL3 (active ingredient: sodium lauryl diethoxy sulfate)/sheep daily for 3 d with 2 d of fasting. Treatment 1 (control) involved feeding faunated sheep a diet in which the supplemental N (45% of total dietary N) was 67% SBM N and 33% urea N. Treatment 2 involved feeding defaunated sheep the same diet as the control. Treatments 3, 4, and 5 involved feeding defaunated sheep diets in which the supplemental N source was either 67% CGM-BM (1:1 N ratio) N:33% urea N, or 33% CGM-BM N:67% urea N or 33% CGM-BM N:33% urea N:33% casein N, respectively. Compared with the faunated control, defaunation decreased (P less than .05) ruminal ammonia concentration (19 vs 26 mg/dl) and increased (P less than .05) CP flow to the duodenum (253 vs 214 g/d) due to a trend for increases in both bacterial (BCP) and nonbacterial (NBCP) CP flows.(ABSTRACT TRUNCATED AT 250 WORDS)