Effects of increasing levels of supplemental protein and naloxone injections on ruminal fermentation, serum hormones and metabolites in lambs were studied in two trials. Supplement × naloxone and sampling time × treatment interactions (Trial 1) for all variables were not significant. Serum growth hormone (GH) decreased (P<0.05) with supplemental protein (4.4 for supplemented lambs vs. 7.1 ng/ml for controls). Serum insulin (INS) was increased (P<0.01) by supplementation, but naloxone did not affect GH or INS. Supplementation elevated (P<0.01) serum urea N and reduced (P<0.01) serum free fatty acid (FFA) concentrations but did not affect serum glucose. Naloxone injections did not affect (P>0.10) concentrations of serum metabolites. Protein supplementation increased (P<0.04) ruminal ammonia concentrations (Trial 2), but ruminal pH did not change as protein level increased. Ruminal proportions of propionate (P<0.03) responded cubically to supplemental protein level. Serum GH changed quadratically (P<0.01) with increasing protein, and serum INS rose linearly (P<0.05) with protein level. Serum glucose and FFA were not affected by supplemental protein (Trial 2). Serum urea N increased (P<0.04) with protein, especially when protein was fed at 125 and 150% of requirements. Supplemental protein has a positive effect on ruminal fermentation of low-quality roughage by sheep and alters metabolic hormone profiles in a manner reflecting an improved protein and energy status.